Method and compositions for efficient and specific rolling circle amplification

Abstract

Disclosed are compositions and methods for nucleic acid amplification reactions that reduce, prevent, or eliminate artifacts; increase efficiency; increase specificity; and/or increase consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of copending application Ser. No. 09/803,713, filed Mar. 9, 2001, entitled “Open Circle Probes With Intramolecular Stem Structures,” by Osama A. Alsmadi and Patricio Abarzúa, which application is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is in the field of nucleic acid amplification, and specifically in the area of rolling circle amplification reactions having increased efficiency and specificity.

BACKGROUND OF THE INVENTION

[0003] Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA)(referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA)(U.S. Pat. No. 5,854,033; PCT Application No. WO 97/19193; Lizardi et al., Nature Genetics 19(3):225-232 (1998)); multiple displacement amplification (MDA)(PCT Application WO 99/18241); strand displacement amplification (SDA)(Walker et al., Nucleic Acids Research 20:1691-1696 (1992), Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)); polymerase chain reaction (PCR) and other exponential amplification techniques involving thermal cycling, self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and amplification with QP replicase (Birkenmeyer and Mushahwar, J. Virological Methods 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)); and various linear amplification techniques involving thermal cycling such as cycle sequencing (Craxton et al., Methods Companion Methods in Enzymology 3:20-26 (1991)).

[0004] Rolling Circle Amplification (RCA) driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (Lizardi et al., Nature Genet. 19: 225-232 (1998); U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193). If a single primer is used, RCA generates in a few minutes a linear chain of hundreds or thousands of tandemly-linked DNA copies of a target that is covalently linked to that target. Generation of a linear amplification product permits both spatial resolution and accurate quantitation of a target. DNA generated by RCA can be labeled with fluorescent oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequences. RCA can be used with fluorophore combinations designed for multiparametric color coding (PCT Application No. WO 97/19193), thereby markedly increasing the number of targets that can be analyzed simultaneously. RCA technologies can be used in solution, in situ and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules (Lizardi et al., 1998). Ligation-mediated Rolling Circle Amplification (LM-RCA) involves circularization of a probe molecule hybridized to a ′ target sequence and subsequent rolling circle amplification of the circular probe (U.S. Pat. Nos. 5, 854,033 and 6,143,495; PCT Application No. WO 97/19193).

[0005] Artifacts—that is, unwanted, unexpected, or non-specific nucleic acid molecules—have been observed in almost all nucleic acid amplification reactions. For example, Stump et al., Nucleic Acids Research 27:4642-4648 (1999), describes nucleic acid artifacts resulting from an illegitimate PCR process during cycle sequencing. In rolling circle amplification, uncircularized open circle probes could prime synthesis during amplification of circularized open circle probes. Other forms of artifacts can occur in other types of nucleic acid amplification techniques.

BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed are compositions and methods for nucleic acid amplification reactions that reduce, prevent, or eliminate artifacts; increase efficiency; increase specificity;

[0007] and/or increase consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

[0008] Disclosed are compositions and methods for reducing or eliminating generation of unwanted, undesirable, or non-specific amplification products in nucleic acid amplification reactions. One form of composition is an open circle probe that can form an intramolecular stem structure, such as a hairpin structure, at one or both ends. Open circle probes are useful in rolling circle amplification techniques. The stem structure allows the open circle probe to be circularized when hybridized to a legitimate target sequence but results in inactivation of uncircularized open circle probes. This inactivation, which can involve stabilization of the stem structure, extension of the end of the open circle probe, or both, reduces or eliminates the ability of the open circle probe to prime nucleic acid synthesis or to serve as a template for rolling circle amplification.

[0009] Disclosed are compositions and methods for increasing the efficiency of nucleic acid amplification reactions. Increased efficiency can include, for example, increased amplification and/or signal generation in less time, from less starting material, and/or from less reagents; and/or signal detection during the amplification reaction. One form of method for increasing efficiency is the use of a detection primer, such as a detection rolling circle replication primer. The detection primer produces a signal during amplification as a quenching moiety in or on the primer becomes separated from a fluorescent label on the primer. A useful form of detection primer is a detection rolling circle primer associated with a peptide nucleic acid quencher. The peptide nucleic acid quencher is displaced from the detection primer as amplification proceeds (via, for example, replication of a nucleic acid strand complementary to the nucleic acid strand that incorporates the primer).

[0010] Another form of method for increasing efficiency is the use of combinations of primers having different relationships to open circle probes used in the method. For example, the use of two or more rolling circle replication primers and one or more secondary DNA strand displacement primers, with each primer specific for a different sequence or region of the open circle probes, can increase the efficiency of amplification by producing multiple simultaneous initiations of replication and multiple simultaneous generations of amplification product simultaneously. For example, each of two or more different rolling circle replication primers can simultaneously prime replication from different sequences in a given circularized open circle probe or amplification target circle. This multiplies the yield of amplification. Use of both rolling circle replication primers (which prime replication of circularized open circle probes and amplification target circles) and secondary DNA strand displacement primers (which prime replication of the product of replication of circularized open circle probes and amplification target circles) allows multiple generations of amplification product to be generated simultaneously. This multiplies the yield of amplification.

[0011] Disclosed are compositions and methods for increasing the specificity of nucleic acid amplification reactions. Increased specificity can include, for example, more amplification of amplification targets, or more amplification based on specific targets, relative to non-target amplification and/or more accurate assessment of false positive and false negative amplification. One form of method for increasing specificity is the use of matched open circle probe sets. Matched open circle probes are open circle probes that are targeted to different forms of the same target sequence. For example, a target sequence in a gene of interest may occur in two or more forms (for example, a “wild type” or “normal” form and a “mutant” form; or, more generally, polymorphic forms); single nucleotide polymorphisms are an example of such different forms of target sequences. By targeting two or more (up to, for example, most or all) of the different forms of a target sequence that may be present, the amplification reaction will include a positive control. That is, for example, the open circle probe targeted to the normal form of the target sequence will produce a signal even if the mutant form of the target sequence is not present in the reaction or the open circle probe targeted to the mutant form of the target sequence will produce a signal even if the normal form of the target sequence is not present in the reaction.

[0012] Disclosed are compositions and methods for increasing the consistency of nucleic acid amplification reactions. Increased consistency can include, for example, levels of amplification products that more accurately reflect the relative amount of starting material, and/or less variation in the yield of amplification from different amplification targets. One form of method for increasing consistency involves the use of three primers having different relationships to open circle probes used in the method. The three primers are detection rolling circle replication primers, secondary DNA strand displacement primers, and common rolling circle replication primers. For example, for a given set of open circle probes or amplification target circles, detection rolling circle replication primers can each correspond to a different open circle probe or amplification target circle in the set while secondary DNA strand displacement primers and common rolling circle replication primers can correspond to all of the open circle probe or amplification target circles in the set. These relationships allow the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of two of the primers used (and their complements on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency). Differential detection is mediated by the circle-specific detection rolling circle replication primers.

[0013] Disclosed are compositions and methods for nucleic acid amplification reactions that involve or produce a combination of the above effects. That is, nucleic acid amplification reactions can combine two or more of reduction, prevention, or elimination of artifacts; increased efficiency; increased specificity; and/or increased consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

[0014] The disclosed open circle probes can be inactivated in several ways. For example, where the 3′ end of an open circle probe is involved in an intramolecular stem structure, the 3′ end can be extended in a replication reaction using the open circle probe sequences as template. Stabilization of the stem structure results in a reduction or elimination of the ability of the open circle probe to prime nucleic acid synthesis because the 3′ end is stably hybridized to sequences in the open circle probe under the conditions used for nucleic acid replication. The open circle probe can also be inactivated by formation of the intramolecular stem structure during the amplification reaction. As long as the end remains in the intramolecular stem structure, it is not available for priming nucleic acid synthesis. A useful form of open circle probe includes a loop as part of the intramolecular stem structure. Hybridization of the loop to the target sequence disrupts the intramolecular stem structure while hybridization of the loop to a mismatched or non-target sequence will not. Thus, the sequence-discrimination ability of the open circle probe determines inactivation of the open circle probe. A hybridization nucleating loop can also be used in linear primers used for nucleic acid replication and amplification.

[0015] The disclosed method is useful for detection, quantitation, and/or location of any desired analyte, such as proteins and peptides. The disclosed method can be multiplexed to detect numerous different analytes simultaneously or used in a single assay. Thus, the disclosed method is useful for detecting, assessing, quantitating, profiling, and/or cataloging gene expression and the presence of nucleic acids and protein in biological samples. The disclosed method is also particularly useful for detecting and discriminating single nucleotide differences in nucleic acid sequences. Thus, the disclosed method is useful for extensive multiplexing of target sequences for sensitive and specific detection of the target sequences themselves or analytes to which the target sequences have been associated. The disclosed method is applicable to numerous areas including, but not limited to, analysis of proteins present in a sample (for example, proteomics analysis), disease detection, mutation detection, protein expression profiling, RNA expression profiling, gene discovery, gene mapping (molecular haplotyping), agricultural research, and virus detection.

[0016] It is an object of the present invention to provide a method of reducing, preventing, or eliminating artifacts in nucleic acid amplification reactions.

[0017] It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.

[0018] It is another object of the present invention to provide kits for nucleic acid amplification that can reduce, prevent, or eliminate artifacts in the nucleic acid amplification reaction.

[0019] It is another object of the present invention to provide a more efficient method of nucleic acid amplification.

[0020] It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, produce a more efficient nucleic acid amplification.

[0021] It is another object of the present invention to provide kits for nucleic acid amplification that produce a more efficient nucleic acid amplification.

[0022] It is another object of the present invention to provide a more specific method of nucleic acid amplification.

[0023] It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, produce a more specific nucleic acid amplification.

[0024] It is another object of the present invention to provide kits for nucleic acid amplification that produce a more specific nucleic acid amplification.

[0025] It is another object of the present invention to provide a more consistent method of nucleic acid amplification.

[0026] It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, produce a more consistent nucleic acid amplification.

[0027] It is another object of the present invention to provide kits for nucleic acid amplification that produce a more consistent nucleic acid amplification.

[0028] It is another object of the present invention to provide a method of nucleic acid amplification that, in combination, reduces, prevents, or eliminates artifacts, is more efficient, is more specific, and/or is more consistent.

[0029] It is another object of the present invention to provide open circle probes and primers that, when used in a nucleic acid amplification reaction, produce a nucleic acid amplification that, in combination, reduces, prevents, or eliminates artifacts, is more efficient, is more specific, and/or is more consistent.

[0030] It is another object of the present invention to provide kits for nucleic acid amplification that produce a nucleic acid amplification that, in combination, reduces, prevents, or eliminates artifacts, is more efficient, is more specific, and/or is more consistent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a diagram illustrating an open circle probe that forms hairpin intramolecular stem structures at both ends (top left). The open circle probe is shown hybridized to a target sequence and ligated (top right). Possible intramolecular structures formed by the ligated open circle probe are also shown (bottom).

[0032] FIGS. 2A and 2B are diagrams illustrating an open circle probe that forms a stem and loop intramolecular stem structure. If the target sequence is present, the open circle probe will hybridize to the target sequence, be ligated, and serve as a template in rolling circle amplification (FIG. 2A). If the target sequence is not present, the intramolecular structure remains and the 3′ end of the open circle probe is extended using the “other” strand as template (FIG. 2B).

[0033] FIG. 3 is a diagram illustrating hybridization, ligation, and amplification of an open circle probe that forms a stem and loop intramolecular stem structure. Hybridization to the target sequence is nucleated by interaction between nucleotides in the loop of the open circle probe and nucleotides in the target sequence (left). This nucleation causes the intramolecular stem structure to be disrupted (middle bottom). The freed end can now hybridize to the target sequence, adjacent to the other end of the probe (right bottom). The open circle probe can then be ligated, thus circularizing the probe, followed by rolling circle amplification of the circularized probe (right top).

[0034] FIGS. 4A, 4B, and 4C are diagrams illustrating hybridization of an open circle probe that forms a stem and loop intramolecular stem structure to a non-target sequence. In most cases, hybridization of loop sequences to a non-target sequence will leave the intramolecular stem structure intact (FIG. 4B). The open circle probe will not be circularized. Even if hybridization of the loop to a non-target sequence were to disrupt the intramolecular stem structure, the non-target sequence is unlikely to have nucleotides complementary to end sequences of the open circle probe (FIG. 4C).

[0035] FIG. 5 is a graph of end point fluorescent signal for mutant targets versus end point fluorescent signal for wild type targets. This is an X-Y plot of end point fluorescent readings obtained from the samples in FIGS. 6A-6F. The X-axis shows Cy3 fluorescence (arbitrary units) corresponding to the mutant genotype. The Y-axis shows FAM signal corresponding to wild type genotype, also in arbitrary units. End point readings fall into three clusters that are easily differentiated by genotype, as indicated in the Figure.

[0036] FIGS. 6A, 6B, 6C, 6D, 6E, and 6F depict graphs of fluorescence over time during the course of rolling circle amplification reactions using a matched open circle probe set. The reaction used a FAM labeled detection rolling circle replication primer specific for an open circle probe targeted to the wild type sequence of Factor II prothrombin and a Cy3 labeled detection rolling circle replication primer specific for an open circle probe targeted to the mutant sequence of Factor II prothrombin. FIG. 6A shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single normal human sample. FIG. 6B shows Cy3 fluorescence from the same 32 samples in FIG. 6A. FIG. 6C shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single heterozygous human sample. FIG. 6D shows Cy3 fluorescence from the same 32 samples in FIG. 6C. FIG. 6E shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single homozygous mutant human sample. FIG. 6F shows Cy3 fluorescence from the same 32 samples in FIG. 6E.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Disclosed are compositions and methods for nucleic acid amplification reactions that reduce, prevent, or eliminate artifacts; increase efficiency; increase specificity; and/or increase consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

[0038] Disclosed are compositions and methods for increasing the efficiency of nucleic acid amplification reactions. Increased efficiency can include, for example, increased amplification and/or signal generation in less time, from less starting material, and/or from less reagents; and/or signal detection during the amplification reaction. One form of method for increasing efficiency is the use of a detection primer, such as a detection rolling circle replication primer. The detection primer produces a signal during amplification as a quenching moiety in or on the primer becomes separated from a fluorescent label on the primer. A useful form of detection primer is a detection rolling circle primer associated with a peptide nucleic acid quencher. The peptide nucleic acid quencher is displaced from the detection primer as amplification proceeds (via, for example, replication of a nucleic acid strand complementary to the nucleic acid strand that incorporates the primer).

[0039] The progress of rolling circle amplification reactions can be monitored in real-time (that is, during the reaction) by using detection primers in the amplification. The detection primer produces a signal during amplification as a quenching moiety in or on the primer becomes separated from a fluorescent label on the primer. When a quenching moiety is in proximity to a fluorescent molecule or label, fluorescence is quenched by transfer of energy to the quenching moiety. Fluorescence is detectable once the quenching moiety is no longer in proximity to the fluorescent label. Detection primers are incorporated into amplification products as they prime replication. In the disclosed amplification reactions, the incorporated primer goes on to serve as a template sequence when the nucleic acid strand in which it is incorporated is replicated. A quenching moiety can be placed in proximity to a fluorescent label on the primer, for example, via hybridization of a nucleic acid sequence to which the quenching moiety is attached to sequence of the primer adjacent to the fluorescent label. When the incorporated primer is replicated, the hybrid is disrupted and the quencher moiety is separated from the fluorescent label, which can then produce a fluorescent signal. Thus, as the amplification reaction proceeds, more and more incorporated detection primers are replicated, producing an ever-increasing fluorescent signal that can be monitored as the reaction proceeds.

[0040] Another form of method for increasing efficiency is the use of combinations of primers having different relationships to open circle probes used in the method. For example, the use of two or more rolling circle replication primers and one or more secondary DNA strand displacement primers, with each primer specific for a different sequence or region of the open circle probes, can increase the efficiency of amplification by producing multiple simultaneous initiations of replication and multiple simultaneous generations of amplification product simultaneously. For example, each of two or more different rolling circle replication primers can simultaneously prime replication from different sequences in a given circularized open circle probe or amplification target circle. This multiplies the yield of amplification.

[0041] Rolling circle amplification involves rolling circle replication of a circular template, such as a circularized open circle probe or an amplification target circle. Rolling circle replication can be mediated by a primer, referred to as a rolling circle replication primer, that hybridizes anywhere on the circular temple. Multiple strands can be produced simultaneously by using two or more rolling circle replication primers that hybridize to different sequences (that is, at different locations) in the circular template. Thus, the disclosed method can be performed using of two or more rolling circle replication primers targeted to different sequences in the circular templates. Particularly useful are the use of detection rolling circle replication primers and common rolling circle replication primers in amplification reactions where both a detection rolling circle replication primer and a common rolling circle replication primer correspond to each open circle probe or amplification target circle.

[0042] Use of both rolling circle replication primers (which prime replication of circularized open circle probes and amplification target circles) and secondary DNA strand displacement primers (which prime replication of the product of replication of circularized open circle probes and amplification target circles) allows multiple generations of amplification product to be generated simultaneously. This multiplies the yield of amplification.

[0043] Rolling circle replication of a circular template produces long strands of DNA containing tandem repeats of sequence complementary to the sequence of the circular template. These strands are referred to as tandem sequence DNA. The speed and yield of rolling circle amplification reactions can be greatly increased by replicating the tandem sequence DNA during rolling circle replication. This can be accomplished by using one or more primers complementary to sequence in the tandem sequence DNA. Such primers, referred to as secondary DNA strand displacement primers, have sequence matching sequence in an open circle probe or amplification target circle (and thus are complementary to the tandem sequence DNA). Replication of the tandem sequence DNA produces more nucleic acid, referred to as secondary tandem sequence DNA, and provides a template for further replication by the rolling circle replication primers (which are complementary to sequences in the secondary tandem sequence DNA). These, and subsequent replication products are similarly replicated producing an overall cascade of replication, referred to as exponential rolling circle amplification, that produces a huge amplification in a short time.

[0044] Disclosed are compositions and methods for increasing the specificity of nucleic acid amplification reactions. Increased specificity can include, for example, more amplification of amplification targets, or more amplification based on specific targets, relative to non-target amplification and/or more accurate assessment of false positive and false negative amplification. One form of method for increasing specificity is the use of matched open circle probe sets. Matched open circle probes are open circle probes that are targeted to different forms of the same target sequence. For example, a target sequence in a gene of interest may occur in two or more forms (for example, a “wild type” or “normal” form and a “mutant” form; or, more generally, polymorphic forms); single nucleotide polymorphisms are an example of such different forms of target sequences. By targeting two or more (up to, for example, most or all) of the different forms of a target sequence that may be present, the amplification reaction will include a positive control. That is, for example, the open circle probe targeted to the normal form of the target sequence will produce a signal even if the mutant form of the target sequence is not present in the reaction or the open circle probe targeted to the mutant form of the target sequence will produce a signal even if the normal form of the target sequence is not present in the reaction.

[0045] Ligation-mediated rolling circle amplification should produce amplification products from a given open circle probe when the target sequence of that open circle probe is present and should not produce amplification products from that open circle probe when the target sequence of that open circle probe is not present. However, it is possible that the absence of the amplification products could be the result of a non-functional reaction rather than the absence of target sequence. Including open target circles specific for two or more possible forms of a target sequence means that the target for at least one of the open circle probes will be present. Resultant production of amplification products serves as a sort of positive control, indicating that the amplification reaction is functional. Further, if there is no target sequence present in the reaction (so that no open circle probe should be circularized and amplified), there is an increased tendency for the reaction to produce spurious or artifactual amplification products. This can be referred to as idle assay artifact production. By ensuring (or increasing the chances) that the target sequence for at least one open circle is present in the amplification reaction, the chance that idle assay artifacts will be produced is minimized.

[0046] Disclosed are compositions and methods for increasing the consistency of nucleic acid amplification reactions. Increased consistency can include, for example, levels of amplification products that more accurately reflect the relative amount of starting material, and/or less variation in the yield of amplification from different amplification targets. One form of method for increasing consistency involves the use of three primers having different relationships to open circle probes used in the method. The three primers are detection rolling circle replication primers, secondary DNA strand displacement primers, and common rolling circle replication primers. For example, for a given set of open circle probes or amplification target circles, detection rolling circle replication primers can each correspond to a different open circle probe or amplification target circle in the set while secondary DNA strand displacement primers and common rolling circle replication primers can correspond to all of the open circle probe or amplification target circles in the set. These relationships allow the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of two of the primers used (and their complements on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency). Differential detection is mediated by the circle-specific detection rolling circle replication primers.

[0047] Rolling circle amplification can be performed using multiple open circle probes or amplification target circles in the same reaction. Specificity of detection of rolling circle replication of different circularized open circle probes and amplification target circles can be accomplished in numerous ways. For real-time detection, it is useful to use a different detection rolling circle replication primer specific for each different open circle probe and amplification target circle. Because the different detection rolling circle replication primers may have different priming efficiencies (due to sequence differences, for example), it is useful to include one or more common rolling circle replication primers that are complementary to all of the open circle probes or amplification target circles in the reaction. This provides rolling circle replication unbiased by differing priming efficiencies.

[0048] Disclosed are compositions and methods for reducing or eliminating generation of unwanted, undesirable, or non-specific amplification products in nucleic acid amplification reactions. One form of composition is an open circle probe that can form an intramolecular stem structure, such as a hairpin structure, at one or both ends. Open circle probes are useful in rolling circle amplification techniques. The stem structure allows the open circle probe to be circularized when hybridized to a legitimate target sequence but results in inactivation of uncircularized open circle probes. This inactivation, which usefully involves stabilization of the stem structure, extension of the end of the open circle probe, or both, reduces or eliminates the ability of the open circle probe to prime nucleic acid synthesis or to serve as a template for rolling circle amplification.

[0049] In ligation-mediated rolling circle amplification (LM-RCA), a linear DNA molecule, referred to as an open circle probe or padlock probe, hybridizes to a target sequence and is circularized. The circularized probe is then amplified via rolling circle replication of the circular probe. Uncircularized probe that remains in the reaction can hybridize to nucleic acid sequences in the reaction and cause amplification of undesirable, non-specific sequences. The disclosed compositions and method address this problem by reducing or eliminating the potential uncircularized open circle probes from priming nucleic acid synthesis.

[0050] The disclosed open circle probes can be inactivated in several ways. For example, where the 3′ end of an open circle probe is involved in an intramolecular stem structure, the 3′ end can be extended in a replication reaction using the open circle probe sequences as template (see FIG. 2B). The result is stabilization of the intramolecular stem structure and a change in the 3′ end sequence. Stabilization of the stem structure results in a reduction or elimination of the ability of the open circle probe to prime nucleic acid synthesis because the 3′ end is stably hybridized to sequences in the open circle probe under the conditions used for nucleic acid replication.

[0051] The open circle probe can also be inactivated by formation of the intramolecular stem structure during the amplification reaction. As long as the end remains in the intramolecular stem structure, it is not available for priming nucleic acid synthesis. This form of inactivation is aided by design the intramolecular stem structure, or selecting amplification conditions, such that the intramolecular hybrid remains stable during rolling circle amplification.

[0052] One form of the disclosed open circle probes includes a loop as part of the intramolecular stem structure. It is useful for the loop to contain sequences complementary to the target sequence. This allows the loop to nucleate hybridization of the open probe to the target sequence. Useful forms of the loop-containing probes are characterized by a sequence discrimination capability that is markedly better that the comparable linear probes due to the competition between the structural interferences between folding due to intramolecular stem formation and linear rigidity due to hybridization of the probe sequence to the target (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). Useful open circle probes of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end(s) of the open circle probe involved in an intramolecular stem structure unavailable for ligation to circularize the probe and leave the 3′ end available for inactivating extension. The presence of target sequence causes the correctly matched open circle probe to unfold, allowing the ends to hybridize to the target sequence and be coupled (see FIG. 3). Where sequences in the loop nucleate hybridization of the open circle probe to a target sequence, loop hybridization to a non-target sequence is unlikely to lead to circularization of the open circle probe. This is because it is unlikely that a non-target sequence will include adjacent sequences to which both the loop and open circle probe end can hybridize (see FIG. 4).

[0053] A hybridization nucleating loop can also be used in linear primers used for nucleic acid replication and amplification. Such a primer forms an intramolecular stem structure, including a loop. Loop-containing primers of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end of the primer involved in an intramolecular stem structure unavailable for priming. The legitimate primer complement sequence causes the correctly matched primer to unfold, allowing the end to hybridize to the primer complement sequence and prime synthesis. Where sequences in the loop nucleate hybridization of the primer, loop hybridization to an illegitimate sequence is unlikely to lead to priming. This is because it is unlikely that an illegitimate sequence will include adjacent sequences to which both the loop and the primer end can hybridize. Including proximity-sensitive labels used in molecular beacon probes in such primers allows hybridization and priming by the primers to be detected through activation of the label upon disruption of the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)).

[0054] Disclosed are compositions and methods for nucleic acid amplification reactions that involve or produce a combination of the above effects. That is, nucleic acid amplification reactions can combine two or more of reduction, prevention, or elimination of artifacts; increased efficiency; increased specificity; and/or increased consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

[0055] The disclosed method is useful for detection, quantitation, and/or location of any desired analyte. The disclosed method can be multiplexed to detect numerous different analytes simultaneously or used in a single assay. Thus, the disclosed method is useful for detecting, assessing, quantitating, profiling, and/or cataloging gene expression and the presence of protein in biological samples. The disclosed method is also particularly useful for detecting and discriminating single nucleotide differences in nucleic acid sequences. This specificity is possible due to the sensitivity of the intramolecular stem structure in loop-containing probes and primers to mismatches between the loop sequence and a prospective target sequence. Thus, the disclosed method is useful for extensive multiplexing of target sequences for sensitive and specific detection of the target sequences themselves or analytes to which the target sequences have been associated. The disclosed method is also useful for detecting, assessing, quantitating, and/or cataloging single nucleotide polymorphisms, and other sequence differences between nucleic acids, nucleic acid samples, and sources of nucleic acid samples.

[0056] The disclosed method is useful for detecting any desired sequence or other analyte, such as proteins and peptides. In particular, the disclosed method can be used to localize or amplify signal from any desired analyte. For example, the disclosed method can be used to assay tissue, transgenic cells, bacterial or yeast colonies, cellular material (for example, whole cells, proteins, DNA fibers, interphase nuclei, or metaphase chromosomes on slides, arrayed genomic DNA, RNA), and samples and extracts from any of biological source. Where target sequences are associated with an analyte, different target sequences, and thus different analytes, can be sensitively distinguished. Specificity of such detection is aided by sensitivity of a loop in an open circle probe to mismatches.

[0057] The disclosed method is applicable to numerous areas including, but not limited to, analysis of proteins present in a sample (for example, proteomics analysis), disease detection, mutation detection, protein expression profiling, RNA expression profiling, gene discovery, gene mapping (molecular haplotyping), agricultural research, and virus detection. Notable uses include protein and peptide detection in situ in cells, on microarrays, protein expression profiling; mutation detection; detection of abnormal proteins or peptides (for example, overexpression of an oncogene protein or absence of expression of a tumor suppressor protein); expression in cancer cells; detection of viral proteins in cells; viral protein expression; detection of inherited diseases such as cystic fibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia; assessment of predisposition for cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, testicular cancer, pancreatic cancer. The disclosed method can also be used for detection of nucleic acids in situ in cells, on microarrays, on DNA fibers, and on genomic DNA arrays; detection of RNA in cells; RNA expression profiling; molecular haplotyping; mutation detection; detection of abnormal RNA (for example, overexpression of an oncogene or absence of expression of a tumor suppressor gene); expression in cancer cells; detection of viral genome in cells; viral RNA expression; detection of inherited diseases such as cystic fibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia; assessment of predisposition for cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, testicular cancer, pancreatic cancer.

[0058] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation. The DNA ligation operation can comprise circularization of one or more open circle probes and can be carried out in the presence of a set of open circle probes. The set of open circle probes can comprise a plurality of different open circle probes. Each open circle probe can comprise two ends, where at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure can be dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set.

[0059] The amplification operation can comprise rolling circle replication of the circularized open circle probes. The amplification operation can be carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in the set of open circle probes.

[0060] Some forms of the disclosed method can comprise an amplification operation. The amplification operation can be carried out in the presence of a set of amplification target circles. The set of amplification target circles can comprise a plurality of different amplification target circles. The amplification operation can comprise rolling circle replication of the amplification target circles. The amplification operation can be carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer can correspond to a different amplification target circle in the set of amplification target circles, the secondary DNA strand displacement primer can correspond to all of the amplification target circles in the set of amplification target circles, and the common rolling circle replication primer can correspond to all of the amplification target circles in the set of amplification target circles.

[0061] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes, and where two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0062] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes. The amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, where each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

[0063] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The amplification operation is carried out in the presence of one or more rolling circle replication primers, where at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher.

[0064] Some forms of the disclosed method can comprise an amplification operation, where the amplification operation comprises rolling circle replication of the amplification target circles. The amplification operation is carried out in the presence of one or more rolling circle replication primers, where at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher.

[0065] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and wherein the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes. Each open circle probe comprises two ends, where at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0066] The amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer is associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

[0067] Some forms of the disclosed method can comprise

[0068] (a) mixing a set of open circle probes with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture. The set of open circle probes comprises a plurality of different open circle probes. Each open circle probe can comprise two ends. At least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure can be dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set.

[0069] (b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles. The amplification target circles formed from the open circle probes in the set of open circle probes can comprise a set of amplification target circles.

[0070] (c) mixing a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in the set of open circle probes.

[0071] (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles. Replication of the amplification target circles results in the formation of tandem sequence DNA.

[0072] In some forms of the disclosed method, rolling circle amplification can be performed using peptide nucleic acid (PNA) quenched primers. Amplification primers (such as detection rolling circle replication primers, common rolling circle replication primers and secondary DNA strand displacement primers) for the reaction can contain a fluorescent dye, with the fluorescence quenched by a PNA molecule that incorporates a quencher. In brief, the PNA anneals to the amplification primer in a manner such that the fluor and quencher are adjacent to one another. After primer incorporation of the primer, the PNA is displaced, resulting in an increase in fluorescence. Examples of useful PNA quenchers are available from Boston Probes.

[0073] The disclosed compositions and method can be illustrated with the following example of oligonucleotides. The oligonucleotides have a 5′ phosphate unless a different molecule or moiety is indicated. An example of a peptide nucleic acid quencher (designated Q-PNA-13) having a Dabcyl quencher is:

[0074] Ac-X-OO-TGA-TTG-CGA-ATG-Lys(Dabcyl) (SEQ ID NO:1)

[0075] This peptide nucleic can be annealed to primers, such as the following examples of two detection rolling circle replication primers (primer 1066 PI FAM and primer 5901 P1 Cy 3) and a secondary DNA strand displacement primer (primer 1704 P2). These primers were designed for use with open circle probes targeted to different forms of a Factor V Leiden target sequence (a wild type form and a mutant form), also shown below. The sequence in the rolling circle replication primers that is complementary to the sequence in the peptide nucleic aid quencher is underlined. The complementary portions of the rolling circle replication primers and the primer complement portions of the open circle probes are in bold. The fluorescent moieties (Cy3 and FAM) are shown at the 5′ end of the primers. The secondary DNA strand displacement primer has sequence matching sequence in the open circle probes. This matching sequence in the open circle probes is in italic. In the open circle probes, sequence that forms a stem is underlined and sequence that is in the resulting loop is shown in lowercase letters. The open circle probes constitute a matched open circle probe set because they are targeted to different forms of the same sequence. 1 1066 P1 FAM (sequence 1066): 5′-/6-FAM/TCATTCGCAATCAATG GGCACCGAAGAA-3′ (SEQ ID NO:2) 5901 P1 Cy3 (sequence 5901): 5′-/Cy3/TCATTCGCAATCAACGGCCGATAACAGA-3′ (SEQ ID NO:3) 1704 P2 (sequence 1704): 5′-CGC GCA GAC ACG ATA-3′ (SEQ ID NO:4)

[0076] Factor V Leiden wild type open circle probe (OCP FV 1066/1704-2 wt (78 bases long)): 2 (SEQ ID NO:5) 5′-GCCTGTCCAGGGATCTGCTTCTTCGGTCCCATCGCGCAGACACGATA GAGGAATACAacaaaataccTGTATTCCTC-3′

[0077] This OCP has a 10 nt long loop (in lowercase) and a 10 bp stem sequence (underlined). The Tm of the stem, calculated using Oligo 6, is 64.3° C. The Tm of a perfectly matched 3′-arm (lowercase sequence and 3′ underlined sequence; this is the left target probe portion) is 53.2° C., and the Tm of a 3′-arm containing a single base mismatch is 47.2° C. Tm of the 5′ arm (plain black text; this is the right target probe portion) is 77.2° C. The primer complement portion for primer 1066 P1 FAM is in bold, and the primer matching portion for the secondary DNA strand displacement primer 1704 P2 is in italic.

[0078] Factor V Leiden mutant open circle probe (OCP FV 5901/1704-2 (78 bases long): 5′-GCCTGTCCAGGGATCTGCTCTGTTATCGGCCGTCGCGCAGACACGATA AAGGAATACAacaaaataccTGTATTCCTT-3′ (SEQ ID NO:6)

[0079] This OCP has a 10 nt long loop (in lowercase) and a 10 bp stem sequence (underlined). The Tm of the stem, calculated using Oligo 6, is 64.4° C. The Tm of a perfectly matched 3′ arm (lowercase sequence and 3′ underlined sequence; this is the left target probe portion) is 53.2° C., and the Tm of a 3′-arm containing a single base mismatch is 47.2° C. Tm of the 5′ arm (plain black text; this is the right target probe portion) is 77.2° C. The primer complement portion for primer 5901 P1 Cy3 is in bold, and the primer matching portion for the secondary DNA strand displacement primer 1704 P2 is in italic.

[0080] The assay for both alleles can be performed in a single tube. This offers the advantage of having two probes in one tube, halving the number of experiments required to obtain the genotype of a locus. Two alleles per well also offers the advantage of increased ligation discrimination, because it guarantees that a probe will anneal perfectly to target regardless of the genotype. The presence of a perfectly matched ligation probe reduces the chance of forced misligation due to mass action. Using matched open circle probes reduces the appearance of artifactual synthesis. Because both probes are present, there is always legitimate circular amplification template being created. Further, conditions can be used such that ERCA will out-compete any amplification artifact.

[0081] Both open circle probes in the assay use the same secondary DNA strand displacement primer (primer 1704 P2). Using the same secondary DNA strand displacement primer for multiplexed open circle probes is a general design improvement that reduces the chance for variation in priming efficiency from sequence to sequence, and therefore provides more uniform amplification for each open circle probe.

[0082] No overnight enzymatic digestion of genomic DNA is required. A 10 minute heat step following ligation can be used. For example, the DNA to 90-96° C. (95° C. is preferred) for 10 minutes following the ligation operation. This allows amplification to proceed as though the DNA had been digested with a restriction enzyme. Genomic DNA amplified using multiple displacement amplification can be genotyped directly (that is, without the need for purification) using the disclosed method.

[0083] Because the rolling circle replication primers and secondary DNA strand displacement primers can correspond to arbitrary sequences in open circle probes, the same primers can be used for multiple different open circle probes (for example, open circle probes targeted to different target sequences).

[0084] Multiple rolling circle replication primers and/or multiple secondary DNA strand displacement primers can be used with the same open circle probe. Further, the primers can be the same for multiple open circle probes used in the reaction. This speeds up the amplification and can help prevent biased amplification by eliminating sequence differences (and thus, sequence-based differences in priming efficiency). The sequences of an additional rolling circle replication primer (primer FV P5e; an example of a common rolling circle replication primer) and an additional secondary DNA strand displacement primer (primer FV P3d) for the Factor V Leiden open circle probes are shown below. 3 FV P5e: GATCCCTGGACAGGC (SEQ ID NO:7) FV P3d: GAGGAATACAACAAAATA (SEQ ID NO:8)

[0085] A. Rolling Circle Amplification

[0086] The disclosed probes and primers are generally useful in rolling circle amplification (RCA) reactions. Rolling circle amplification is described in U.S. Pat. Nos. 5,854,033 and 6,143,495. Rolling circle amplification involves amplifying nucleic acid sequences based on the presence of a specific target sequence or analyte, such as a protein or peptide. The method is useful for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. Useful embodiments of the method, referred to as ligation-mediated RCA (LM-RCA), consist of a DNA ligation operation, an amplification operation, and, optionally, a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule (referred to as an open circle probe). This step is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. By coupling a nucleic acid tag to a specific binding molecule, such as an antibody, amplification of the nucleic acid tag can be used to detect analytes in a sample. This is useful for detection of analytes where a target nucleic acid sequence is part of a reporter binding molecule, where an amplification target circle serves as an amplifiable tag on a reporter binding molecule, or where an amplification target circle is amplified using a rolling circle replication primer that is part of a reporter binding molecule. Optionally, an additional amplification operation can be performed on the DNA produced by rolling circle replication. Rolling circle amplification can also be performed independently of a ligation operation.

[0087] During or following amplification, the amplified sequences can be detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Major advantages of this method are that the ligation operation can be manipulated to obtain allelic discrimination and the amplification operation is isothermal. In multiplex assays, the primer oligonucleotide used for DNA replication can be the same for all probes, or subsets of probes can be used for different sets of amplified nucleic acids to be detected. Rolling circle amplification is especially suited to sensitive detection of multiple analytes, such as proteins and peptides, in a single assay, reaction, or assay system.

[0088] Rolling circle amplification has two features that provide simple and consistent amplification and detection of a target nucleic acid sequence. First, target sequences are amplified via a small diagnostic probe with an arbitrary primer binding sequence. This allows consistency in the priming and replication reactions, even between probes having very different target sequences. Second, amplification takes place not in cycles, but in a continuous, isothermal replication: rolling circle replication. This makes amplification less complicated and much more consistent in output.

[0089] The disclosed compositions can also be used in methods for of multiplex detection of molecules of interest involving rolling circle replication. The methods are useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The methods also have an inherently low level of background signal. A useful form of such a method consists of an association operation, an amplification operation, and a detection operation. The method can also include a ligation operation. The association operation involves association of one or more specially designed reporter binding molecules, either wholly or partly nucleic acid, to target molecules of interest. The reporter binding molecules can target any molecule of interest but preferably targets proteins or peptides. This operation associates the reporter binding molecules to a target molecules present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. By coupling a nucleic acid tag to a specific binding molecule, such as an antibody, amplification of the nucleic acid tag can be used to detect analytes in a sample.

[0090] Following rolling circle replication, the amplified sequences can be detected using combinatorial multicolor coding probes (or other multiplex detection system) that allow separate and simultaneous detection of multiple different amplified target sequences representing multiple different target molecules. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. The target molecules can be analytes of any nature (such as proteins and peptides) by associating the target sequences to be amplified with the target molecules.

Materials

[0091] A. Open Circle Probes

[0092] An open circle probe (OCP) is a linear DNA molecule, preferably containing between 50 to 1000 nucleotides, more preferably between about 60 to 150 nucleotides, and most preferably between about 70 to 100 nucleotides. The OCP has a 5′ phosphate group and a 3′ hydroxyl group. This allows the ends to be ligated (to each other or to other nucleic acid ends) using a ligase, coupled, or extended in a gap-filling operation. Useful open circle probes for use in the disclosed method can form an intramolecular stem structure involving one or both of the OCP's ends. Such open circle probes are referred to herein as hairpin open circle probes. An intramolecular stem structure involving an end refers to a stem structure where the terminal nucleotides (that is, nucleotides at the end) of the OCP are hybridized to other nucleotides in the OCP (FIGS. 1 and 2). Open circle probes can be partially double-stranded.

[0093] The intramolecular stem structure can form a hairpin structure or a stem and loop structure. If both ends of an OCP are involved in an intramolecular stem structure, the two ends of the OCP can each form a separate intramolecular stem structure or can together form a single intramolecular stem structure. In the latter case the two ends would be hybridized together. In some forms, the 3′ end of the open circle probe can form an intramolecular stem structure. The 5′ end of the open circle probe can also form an intramolecular stem structure, either alone, or in the same open circle probe having an intramolecular stem structure at the 3′ end. The intramolecular stem structure can form, for example, under conditions suitable for nucleic acid replication, and in particular under conditions used for nucleic acid replication when the open circle probe is being used. For example, the intramolecular stem structure can be designed to form under conditions used for rolling circle replication. The formation of the intramolecular stem structure during replication allows the structure to reduce or prevent participation of uncircularized open circle probes in nucleic acid replication. In particular, the intramolecular stem structure prevents the open circle probe in which the structure forms from serving as a template for rolling circle replication, from priming nucleic acid replication, or both. This follows from the sequestration of the end of uncircularized open circle probe in the stem. The end of the open circle probe cannot hybridize to, and prime from, another sequence while sequestered in the intramolecular stem structure. It is also useful for the intramolecular stem structure to be more stable than hybrids between the open circle probe and mismatched sequences.′ In this way, the intramolecular stem structure will be thermodynamically favored over undesired primer hybridizations. Open circle probes that form intramolecular stem structures at the 3′ end will have the 3′ end extended during replication (using open circle probe sequences as template). This serves to stabilize the intramolecular stem structure in the uncircularized open circle probes, making them unavailable for priming.

[0094] Portions of the OCP can have specific functions making the OCP useful for RCA and LM-RCA. These portions are referred to as the target probe portions, the primer complement portions, the spacer region, the secondary DNA strand displacement primer matching portions, the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portions. The target probe portions and at least one primer complement portion are required elements of an open circle probe. The primer complement portion can be part of, for example, the spacer region. Detection tag portions, secondary target sequence portions, promoter portions, and additional primer complement portions are optional and, when present, can be part of, for example, the spacer region. Address tag portions are optional and, when present, can be part of, for example, the spacer region. The primer complement portions, and the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portions, if present, can be non-overlapping. However, various of these portions can be partially or completely overlapping if desired. OCPs can be single-stranded but may be partially double-stranded. In use, the target probe portions of an OCP should be single-stranded so that they can interact with target sequences. Generally, an open circle probe is a single-stranded, linear DNA molecule comprising, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer region, a left target probe portion, and a 3′ hydroxyl group, with a primer complement portion present as part of the spacer region. Particularly useful open circle probes comprise a right target probe portion, a left target probe portion, a detection primer complement portion, a secondary DNA strand displacement primer matching portion, and a common primer complement portion. Those segments of the spacer region that do not correspond to a specific portion of the OCP can be arbitrarily chosen sequences. It is preferred that OCPs do not have any sequences that are self-complementary. It is considered that this condition is met if there are no complementary regions greater than six nucleotides long without a mismatch or gap. It is also preferred that OCPs containing a promoter portion do not have any sequences that resemble a transcription terminator, such as a run of eight or more thymidine nucleotides.

[0095] The open circle probe, when ligated and replicated, gives rise to a long DNA molecule containing multiple repeats of sequences complementary to the open circle probe. This long DNA molecule is referred to herein as tandem sequences DNA (TS-DNA). TS-DNA contains sequences complementary to the target probe portions, the primer complement portion, the spacer region, and, if present on the open circle probe, the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portion. These sequences in the TS-DNA are referred to as target sequences (which match the original target sequence), primer sequences (which match the sequence of the rolling circle replication primer), spacer sequences (complementary to the spacer region), detection tags, secondary target sequences, address tags, and promoter sequences. The TS-DNA will also have sequence complementary to the matching portion of secondary DNA strand displacement primers. This sequence in the TS-DNA is referred to as the secondary DNA strand displacement primer complement or as the primer complement.

[0096] 1. Sets of Open Circle Probes

[0097] Open circle probes can be used in sets in the disclosed method. Ligation and amplification operations can involve a single reaction or multiple different reactions. Each reaction can use one or more sets of open circle probes. The same or different sets of open circle probes can be used in different reactions. Useful sets of open circle probes can have particular relationships to detection rolling circle replication primers, common rolling circle replication primers, and secondary DNA strand displacement primers. For example, each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in the set of open circle probes. This relationship has advantages discussed elsewhere herein.

[0098] The disclosed method also can be carried out using multiple open circle probe sets. The sets can each include a plurality of different open circle probes. The primer relationships described above can be extended to multiple open circle probe sets. For example, each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in all of the sets of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in all of the sets of open circle probes. As another example, each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes and the common rolling circle replication primer can correspond to all of the open circle probes in all of the sets of open circle probes. The amplification operation can then be carried out in the presence of a plurality of secondary DNA strand displacement primers, where each secondary DNA strand displacement primer can correspond to open circle probes in a different set of open circle probes and a single secondary DNA strand displacement primer can correspond to all of the open circle probes in a given set of open circle probes. As another example, each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes, the secondary DNA strand displacement primer and can correspond to all of the open circle probes in all of the sets of open circle probes. The amplification operation can then be carried out in the presence of a plurality of common rolling circle replication primers, where each common rolling circle replication primer can correspond to open circle probes in a different set of open circle probes and a single common rolling circle replication primer can correspond to all of the open circle probes in a given set of open circle probes.

[0099] As another example, each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes. The amplification operation can then be carried out in the presence of a plurality of secondary DNA strand displacement primers and in the presence of a plurality of common rolling circle replication primers. In the reaction, each secondary DNA strand displacement primer can correspond to open circle probes in a different set of open circle probes, a single secondary DNA strand displacement primer can correspond to all of the open circle probes in a given set of open circle probes, each common rolling circle replication primer can correspond to open circle probes in a different set of open circle probes, and a single common rolling circle replication primer and correspond to all of the open circle probes in a given set of open circle probes.

[0100] As other examples, all of the open circle probes in all of the sets of open circle probes can be different, and/or at least one of the detection rolling circle replication primers can correspond to an open circle probe in each of at least two of the sets of open circle probes. As another example, each detection rolling circle replication primer can correspond to a different open circle probe in a given set of open circle probes. At least one of the detection rolling circle replication primers can correspond to an open circle probe in each of at least two of the sets of open circle probes. Other combinations of sets and relationships of primers to sets and members of the sets are contemplated.

[0101] At least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes can be labeled or detected with the same fluorescent moiety as at least one of the detection rolling circle replication primers corresponding to an open circle probe in a different one of the sets of open circle probes. At least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes can be labeled or detected with the same fluorescent moiety as at least one of the detection rolling circle replication primers in the same set of open circle probes.

[0102] Another form of open circle probe set is a matched open circle probe set. In a matched open circle probe set, the open circle probes can be targeted to different forms of the same target sequence. Different forms of a target sequence refer to sequences that are homologous, analogous, allelic, or otherwise similarly related or derived from a common source sequence but that have some difference in sequence. For example, different forms of the same target sequence can include a “wild type” or “normal” form and one or more “mutant” forms of, for example, the same nucleic acid segment, gene, or gene segment; two or more polymorphic forms of, for example, the same nucleic acid segment, gene, or gene segment; two or more allelic forms of, for example, the same nucleic acid segment, gene, or gene segment; or two or more single nucleotide polymorphisms of, for example, the same nucleic acid segment, gene, or gene segment. These are only examples. The different forms of the same target sequence can be any set of target sequences that have an overall sequence similarity but that are not identical. By targeting two or more different forms of a target sequence that may be present, the amplification reaction can include a positive control. That is, for example, an open circle probe targeted to the normal form of the target sequence will produce a signal even if the mutant form of the target sequence is not present in the reaction or an open circle probe targeted to the mutant form of the target sequence will produce a signal even if the normal form of the target sequence is not present in the reaction.

[0103] The different forms of the same target sequence can comprise, for example, a wild type form of the target sequence and a mutant form of the target sequence; a normal form of the target sequence and a mutant form of the target sequence; a wild type form of the target sequence and two mutant forms of the target sequence; a normal form of the target sequence and two mutant forms of the target sequence; a wild type form of the target sequence and a plurality of mutant forms of the target sequence; a normal form of the target sequence and a plurality of mutant forms of the target sequence; two allelic forms of the target sequence; two polymorphic forms of the target sequence; two single nucleotide polymorphisms of the target sequence; a normal form of the target sequence and a single nucleotide polymorphism of the target sequence; a wild type form of the target sequence and a single nucleotide polymorphism of the target sequence; a plurality of allelic forms of the target sequence; a plurality of polymorphic forms of the target sequence; a plurality of single nucleotide polymorphisms of the target sequence; a normal form of the target sequence and a plurality of single nucleotide polymorphisms of the target sequence; a wild type form of the target sequence and a plurality of single nucleotide polymorphisms of the target sequence; or a combination. These are only examples. Other combinations of forms of target sequences are contemplated.

[0104] The set of open circle probes can include one or a plurality of matched open circle probe sets. Open circle probes in different matched open circle probe sets can be targeted to the same or to different target sequences. Thus, open circle probes in one matched open circle probe set can be targeted to different forms of the same target sequence (for example, a first target sequence) while open circle probes in a different matched open circle probe set can be targeted to different forms of a different target sequence (for example, a second target sequence). The different target sequences can be unrelated or can have some relationship to each other. For example, the different target sequences can be in the same gene. Thus, there can be, for example, open circle probe sets that include more than one matched open circle probe sets where the open circle probe in two or more of the matched open circle probe sets are targeted to different target sequences in the same gene. This is useful, for example, for simultaneously testing for the presence of alternative sequences at a number of different sites in a gene. As another example, different target sequences to which open circle probes in different matched open circle probe sets are targeted can be associated with the same disease or condition. This is useful, for example, for simultaneously testing for the presence of multiple different sequences associated with a disease or condition.

[0105] Matched open circle probe sets can be subsets of other open circle probe sets (such as open circle probe sets having the relationships to primers described above). A set of open circle probes can comprise one or a plurality of matched open circle probe sets. Differential detection of different open circle probes in sets of open circle probes and in sets of matched open circle probes can be accomplished generally as described elsewhere herein. For example, different detection rolling circle replication primers can correspond to different open circle probes in the set and can include or be associated with different fluorescent moieties.

[0106] 2. Target Probe Portions

[0107] There are two target probe portions on each OCP, one at each end of the OCP. The target probe portions can each be any length that supports specific and stable hybridization between the target probes and the target sequence. For this purpose, a length of 10 to 35 nucleotides for each target probe portion is preferred, with target probe portions 15 to 25 nucleotides long being most preferred. The target probe portion at the 3′ end of the OCP is referred to as the left target probe, and the target probe portion at the 5′ end of the OCP is referred to as the right target probe. These target probe portions are also referred to herein as left and right target probes or left and right probes. The target probe portions are complementary to a target nucleic acid sequence.

[0108] The target probe portions are complementary to the target sequence, such that upon hybridization the 5′ end of the right target probe portion and the 3′ end of the left target probe portion are base-paired to adjacent nucleotides in the target sequence, with the objective that they serve as a substrate for ligation.

[0109] Where the open circle probe has an intramolecular stem structure that forms a stem and loop structure, it is useful for a portion of one of the target probe portions of the open circle probe to be in the loop of the stem and loop structure. This portion of the target probe portion in the loop can then hybridize to the target sequence of the open circle probe. Such an arrangement allows design of hairpin open circle probes where the stability of the intramolecular stem structure depends on the presence or absence of the specific target sequence. In particular, an open circle probe that forms a stem and loop structure with a portion of the target probe portion in the loop can be designed so that hybridization of the target probe portion in the loop to the target sequence disrupts the intramolecular stem structure (FIG. 2; Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). In this way, the intramolecular stem structure remains intact in the absence of the target sequence and thus reduces or eliminates the ability of the open circle probe to prime nucleic acid replication (or to serve as a template for rolling circle replication). In the presence of the target sequence, disruption of the intramolecular stem structure allows the end of the open circle probe to hybridize to the target sequence. This hybrid between the target sequence and the end of the open circle probe allows the ends of the open circle probe to come into proximity on the target sequence which in turn allows ligation of the ends (FIG. 3). For this form of hairpin open circle probe, it is useful if hybridization of the loop to a sequence other than the target sequence does not disrupt the intramolecular stem structure. The hybrid between the target sequence and the target probe portion at the end of the open circle probe can be more stable than the intramolecular stem structure. This helps stabilize hybridization of the open circle probe to the target sequence in competition with the intramolecular stem structure.

[0110] Discrimination of open circle probe hybridization also can be accomplished by hybridizing probe to target sequence under conditions that favor only exact sequence matches leaving other open circle probes unhybridized. The unhybridized open circle probes will retain or re-form the intramolecular hybrid and the end of the open circle probe involved in the intramolecular stem structure will be extended during replication.

[0111] In another form of open circle probe, the 5′ end and the 3′ end of the target probe portions may hybridize in such a way that they are separated by a gap space. In this case the 5′ end and the 3′ end of the OCP may only be ligated if one or more additional oligonucleotides, referred to as gap oligonucleotides, are used, or if the gap space is filled during the ligation operation. The gap oligonucleotides hybridize to the target sequence in the gap space to form a continuous probe/target hybrid. The gap space may be any length desired but is generally ten nucleotides or less. It is preferred that the gap space is between about three to ten nucleotides in length, with a gap space of four to eight nucleotides in length being most preferred. Alternatively, a gap space could be filled using a DNA polymerase during the ligation operation. When using such a gap-filling operation, a gap space of three to five nucleotides in length is most preferred. As another alternative, the gap space can be partially bridged by one or more gap oligonucleotides, with the remainder of the gap filled using DNA polymerase.

[0112] 3. Primer Complement Portions

[0113] Primer complement portions are parts of an open circle probe that are complementary to rolling circle replication primers (RCRP). Each OCP preferably has at least two primer complement portions: a detection primer complement portion and a common primer complement portion. This allows rolling circle replication to initiate at multiple sites on ligated OCPs. An OCP can include one or more than one detection primer complement portion, and one or more than one common primer complement portion. A single detection primer complement portion is preferred. However, if multiple detection primer complement portions are present, they can have sequence complementary to the same detection rolling circle replication primer (which is preferred), different detection rolling circle replication primers, or a combination of the same and different detection rolling circle replication primers. If multiple common primer complement portions are present, they can have sequence complementary to the same common rolling circle replication primer (which is preferred), different common rolling circle replication primers, or a combination of the same and different common rolling circle replication primers. A primer complement portion and its cognate primer can have any desired sequence so long as they are complementary to each other. The sequence of the primer complement portion is referred to as the primer complement sequence. The primer complement portion complementary to a detection rolling circle replication primer can be referred to as a detection primer complement portion. The primer complement portion complementary to a common rolling circle replication primer can be referred to as a common primer complement portion. The primer complement sequence of a detection primer complement portion can be referred to as a detection primer complement sequence. The primer complement sequence of a common primer complement portion can be referred to as a common primer complement sequence.

[0114] In general, the sequence of a primer complement can be chosen such that it is not significantly similar to any other portion of the OCP. The primer complement portion can be any length that supports specific and stable hybridization between the primer complement portion and the primer. For this purpose, a length of 10 to 35 nucleotides is preferred, with a primer complement portion 16 to 20 nucleotides long being most preferred. The primer complement portion can be located anywhere on the OCP, such as within the spacer region of an OCP. Primer complement portions can be anywhere on the OCP or circularized OCP. For example, the primer complement portions can be adjacent to the right target probe, with the right target probe portion and the primer complement portion preferably separated by three to ten nucleotides, and most preferably separated by six nucleotides, from the proximate primer complement portion. This location prevents the generation of any other spacer sequences, such as detection tags and secondary target sequences, from unligated open circle probes during DNA replication. A primer complement portion can also be a part of or overlap all or a part of the target probe portions and/or any gap space sequence, if present.

[0115] 4. Secondary DNA Strand Displacement Primer Matching Portions

[0116] Secondary DNA strand displacement primer matching portions are parts of an open circle probe that match sequence in secondary DNA strand displacement primers. The sequence in a secondary DNA strand displacement primer that matches a secondary DNA strand displacement primer matching portion in an OCP is referred to as the matching portion of the secondary DNA strand displacement primer. An OCP can include one or more than one primer matching portion. If multiple primer matching portions are present, they can have sequence matching the same secondary DNA strand displacement primer (which is preferred), different secondary DNA strand displacement primers, or a combination of the same and different secondary DNA strand displacement primers. A single secondary DNA strand displacement primer matching portion is preferred. A primer matching portion and its cognate primer can have any desired sequence so long as they are complementary to each other. The sequence of the primer matching portion can be referred to as the primer matching sequence. More specifically, the sequence of the secondary DNA strand displacement primer matching portion can be referred to as the secondary DNA strand displacement primer matching sequence.

[0117] In general, the sequence of a primer matching portion can be chosen such that it is not significantly similar to any other portion of the OCP. Primer matching portions can overlap with primer complement portions, although it is preferred that they not overlap. The primer matching portion can be any length that supports specific and stable hybridization between the primer complement portion in the resulting TS-DNA and the primer. For this purpose, a length of 10 to 35 nucleotides is preferred, with a primer matching portion 16 to 20 nucleotides long being most preferred. The primer matching portion can be located anywhere on the OCP, such as within the spacer region of an OCP. Primer matching portions can be anywhere on the OCP or circularized OCP.

[0118] 5. Detection Tag Portions

[0119] Detection tag portions are part of the spacer region of an open circle probe. Detection tag portions have sequences matching the sequence of the complementary portion of detection probes. These detection tag portions, when amplified during rolling circle replication, result in TS-DNA having detection tag sequences that are complementary to the complementary portion of detection probes. If present, there may be one, two, three, or more than three detection tag portions on an OCP. For example, an OCP can have two, three or four detection tag portions. Most preferably, an OCP will have three detection tag portions. Generally, it is preferred that an OCP have 60 detection tag portions or less. There is no fundamental limit to the number of detection tag portions that can be present on an OCP except the size of the OCP. When there are multiple detection tag portions, they may have the same sequence or they may have different sequences, with each different sequence complementary to a different detection probe. It is preferred that an OCP contain detection tag portions that have the same sequence such that they are all complementary to a single detection probe. For some multiplex detection methods, it is preferable that OCPs contain up to six detection tag portions and that the detection tag portions have different sequences such that each of the detection tag portions is complementary to a different detection probe. The detection tag portions can each be any length that supports specific and stable hybridization between the detection tags and the detection probe. For this purpose, a length of 10 to 35 nucleotides is preferred, with a detection tag portion 15 to 20 nucleotides long being most preferred. Detection tags are less useful when the method involves real-time detection of amplification via detection rolling circle replication primers.

[0120] 6. Secondary Target Sequence Portions

[0121] Secondary target sequence portions are part of the spacer region of an open circle probe. Secondary target sequence portions have sequences matching the sequence of target probes of a secondary open circle probe. These secondary target sequence portions, when amplified during rolling circle replication, result in TS-DNA having secondary target sequences that are complementary to target probes of a secondary open circle probe. If present, there may be one, two, or more than two secondary target sequence portions on an OCP. It is preferred that an OCP have one or two secondary target sequence portions. Most preferably, an OCP will have one secondary target sequence portion. Generally, it is preferred that an OCP have 50 secondary target sequence portions or less. There is no fundamental limit to the number of secondary target sequence portions that can be present on an OCP except the size of the OCP. When there are multiple secondary target sequence portions, they may have the same sequence or they may have different sequences, with each different sequence complementary to a different secondary OCP. It is preferred that an OCP contain secondary target sequence portions that have the same sequence such that they are all complementary to a single target probe portion of a secondary OCP. The secondary target sequence portions can each be any length that supports specific and stable hybridization between the secondary target sequence and the target sequence probes of its cognate OCP. For this purpose, a length of 20 to 70 nucleotides is preferred, with a secondary target sequence portion 30 to 40 nucleotides long being most preferred. As used herein, a secondary open circle probe is an open circle probe where the target probe portions match or are complementary to secondary target sequences in another open circle probe or an amplification target circle. It is contemplated that a secondary open circle probe can itself contain secondary target sequences that match or are complementary to the target probe portions of another secondary open circle probe. Secondary open circle probes related to each other in this manner are referred to herein as nested open circle probes.

[0122] 7. Address Tag Portions

[0123] The address tag portion is part of either the target probe portions or the spacer region of an open circle probe. The address tag portion has a sequence matching the sequence of the complementary portion of an address probe. This address tag portion, when amplified during rolling circle replication, results in TS-DNA having address tag sequences that are complementary to the complementary portion of address probes. If present, there may be one, or more than one, address tag portions on an OCP. It is preferred that an OCP have one or two address tag portions. Most preferably, an OCP will have one address tag portion. Generally, it is preferred that an OCP have 50 address tag portions or less. There is no fundamental limit to the number of address tag portions that can be present on an OCP except the size of the OCP. When there are multiple address tag portions, they may have the same sequence or they may have different sequences, with each different sequence complementary to a different address probe. It is preferred that an OCP contain address tag portions that have the same sequence such that they are all complementary to a single address probe. Preferably, the address tag portion overlaps all or a portion of the target probe portions, and all of any intervening gap space. Most preferably, the address tag portion overlaps all or a portion of both the left and right target probe portions. The address tag portion can be any length that supports specific and stable hybridization between the address tag and the address probe. For this purpose, a length between 10 and 35 nucleotides long is preferred, with an address tag portion 15 to 20 nucleotides long being most preferred.

[0124] 8. Promoter Portions

[0125] The promoter portion corresponds to the sequence of an RNA polymerase promoter. A promoter portion can be included in an open circle probe so that transcripts can be generated from the OCP or TS-DNA. The sequence of any promoter may be used, but simple promoters for RNA polymerases without complex requirements are preferred. It is also preferred that the promoter is not recognized by any RNA polymerase that may be present in the sample containing the target nucleic acid sequence. Preferably, the promoter portion corresponds to the sequence of a T7 or SP6 RNA polymerase promoter. The T7 and SP6 RNA polymerases are highly specific for particular promoter sequences. Other promoter sequences specific for RNA polymerases with this characteristic would also be preferred. Because promoter sequences are generally recognized by specific RNA polymerases, the cognate polymerase for the promoter portion of the OCP should be used for transcriptional amplification. Numerous promoter sequences are known and any promoter specific for a suitable RNA polymerase can be used. The promoter portion can be located anywhere within the spacer region of an OCP and can be in either orientation. Preferably, the promoter portion is immediately adjacent to the left target probe and is oriented to promote transcription toward the 3′ end of the open circle probe. This orientation results in transcripts that are complementary to TS-DNA, allowing independent detection of TS-DNA and the transcripts, and prevents transcription from interfering with rolling circle replication.

[0126] B. Gap Oligonucleotides

[0127] Gap oligonucleotides are oligonucleotides that are complementary to all or a part of that portion of a target sequence which covers a gap space between the ends of a hybridized open circle probe. Gap oligonucleotides have a phosphate group at their 5′ ends and a hydroxyl group at their 3′ ends. This facilitates ligation of gap oligonucleotides to open circle probes, or to other gap oligonucleotides. The gap space between the ends of a hybridized open circle probe can be filled with a single gap oligonucleotide, or it can be filled with multiple gap oligonucleotides. For example, two 3 nucleotide gap oligonucleotides can be used to fill a six nucleotide gap space, or a three nucleotide gap oligonucleotide and a four nucleotide gap oligonucleotide can be used to fill a seven nucleotide gap space. Gap oligonucleotides are particularly useful for distinguishing between closely related target sequences. For example, multiple gap oligonucleotides can be used to amplify different allelic variants of a target sequence. By placing the region of the target sequence in which the variation occurs in the gap space formed by an open circle probe, a single open circle probe can be used to amplify each of the individual variants by using an appropriate set of gap oligonucleotides.

[0128] C. Amplification Target Circles

[0129] An amplification target circle (ATC) is a circular DNA molecule, preferably containing between 40 to 1000 nucleotides, more preferably between about 50 to 150 nucleotides, and most preferably between about 50 to 100 nucleotides. ATCs are preferably single-stranded but may be partially or fully double-stranded. Portions of ATCs have specific functions making the ATC useful for rolling circle amplification (RCA). These portions are referred to as the primer complement portions, the secondary DNA strand displacement primer matching portions, the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portions. These portions are analogous to similarly-named portions of OCPs and their further description elsewhere herein in the context of OCPs is applicable to the analogous portion in ATCs. At least one primer complement portion is a required element of an amplification target circle. Secondary DNA strand displacement primer matching portions, detection tag portions, secondary target sequence portions, address tag portions, and promoter portions are optional. The primer complement portion, and other primer complement portions, the secondary DNA strand displacement primer matching portions, the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portion, if present, are preferably non-overlapping. However, various of these portions can be partially or completely overlapping if desired. Generally, an amplification target circle is a single-stranded, circular DNA molecule comprising a primer complement portion. Particularly useful amplification target circles comprise a detection primer complement portion, a secondary DNA strand displacement primer matching portion, and a common primer complement portion. Those segments of the ATC that do not correspond to a specific portion of the ATC can be arbitrarily chosen sequences. It is preferred that ATCs do not have any sequences that are self-complementary. It is considered that this condition is met if there are no complementary regions greater than six nucleotides long without a mismatch or gap. It is also preferred that ATCs containing a promoter portion do not have any sequences that resemble a transcription terminator, such as a run of eight or more thymidine nucleotides. Ligated and circularized open circle probes are a type of ATC, and as used herein the term amplification target circle includes ligated open circle probes and circularized open circle probes. An ATC can be used in the same manner as described herein for OCPs that have been ligated or circularized.

[0130] An amplification target circle, when replicated, gives rise to a long DNA molecule containing multiple repeats of sequences complementary to the amplification target circle. This long DNA molecule is referred to herein as tandem sequences DNA (TS-DNA). TS-DNA contains sequences complementary to the primer complement portions and, if present on the amplification target circle, the secondary DNA strand displacement primer matching portions, the detection tag portions, the secondary target sequence portions, the address tag portions, and the promoter portion. These sequences in the TS-DNA are referred to as primer sequences (which match the sequence of the rolling circle replication primer), spacer sequences (complementary to the spacer region), detection tags, secondary target sequences, address tags, and promoter sequences. The TS-DNA will also have sequence complementary to the matching portion of secondary DNA strand displacement primers. This sequence in the TS-DNA is referred to as the secondary DNA strand displacement primer complement or as the primer complement. Amplification target circles are useful as tags for specific binding molecules.

[0131] 1. Sets of Amplification Target Circles

[0132] Amplification target circles can be used in sets in the disclosed method. Amplification operations can involve a single reaction or multiple different reactions. Each reaction can use one or more sets of amplification target circles. The same or different sets of amplification target circles can be used in different reactions. Useful sets of amplification target circles can have particular relationships to detection rolling circle replication primers, common rolling circle replication primers, and secondary DNA strand displacement primers. Generally, these are the same relationships that sets of open circle probes can have to the primers, as described elsewhere herein. For example, each detection rolling circle replication primer can correspond to a different amplification target circle in the set of amplification target circles, the secondary DNA strand displacement primer can correspond to all of the amplification target circles in the set of amplification target circles, and the common rolling circle replication primer can correspond to all of the amplification target circles in the set of amplification target circles. This relationship has advantages discussed elsewhere herein.

[0133] The disclosed method also can be carried out using multiple amplification target circle sets. The sets can each include a plurality of different amplification target circles. The primer relationships described above can be extended to multiple amplification target circle sets. For example, each detection rolling circle replication primer can correspond to a different amplification target circle in all of the sets of amplification target circles, the secondary DNA strand displacement primer can correspond to all of the amplification target circles in all of the sets of amplification target circles, and the common rolling circle replication primer can correspond to all of the amplification target circles in all of the sets of amplification target circles. As another example, each detection rolling circle replication primer can correspond to a different amplification target circle in all of the sets of amplification target circles and the common rolling circle replication primer can correspond to all of the amplification target circles in all of the sets of amplification target circles. The amplification operation can then be carried out in the presence of a plurality of secondary DNA strand displacement primers, where each secondary DNA strand displacement primer can correspond to amplification target circles in a different set of amplification target circles and a single secondary DNA strand displacement primer can correspond to all of the amplification target circles in a given set of amplification target circles. As another example, each detection rolling circle replication primer can correspond to a different amplification target circle in all of the sets of amplification target circles, the secondary DNA strand displacement primer and can correspond to all of the amplification target circles in all of the sets of amplification target circles. The amplification operation can then be carried out in the presence of a plurality of common rolling circle replication primers, where each common rolling circle replication primer can correspond to amplification target circles in a different set of amplification target circles and a single common rolling circle replication primer can correspond to all of the amplification target circles in a given set of amplification target circles.

[0134] As another example, each detection rolling circle replication primer can correspond to a different amplification target circle in all of the sets of amplification target circles. The amplification operation can then be carried out in the presence of a plurality of secondary DNA strand displacement primers and in the presence of a plurality of common rolling circle replication primers. In the reaction, each secondary DNA strand displacement primer can correspond to amplification target circles in a different set of amplification target circles, a single secondary DNA strand displacement primer can correspond to all of the amplification target circles in a given set of amplification target circles, each common rolling circle replication primer can correspond to amplification target circles in a different set of amplification target circles, and a single common rolling circle replication primer and correspond to all of the amplification target circles in a given set of amplification target circles.

[0135] As other examples, all of the amplification target circles in all of the sets of amplification target circles can be different, and/or at least one of the detection rolling circle replication primers can correspond to an amplification target circle in each of at least two of the sets of amplification target circles. As another example, each detection rolling circle replication primer can correspond to a different amplification target circle in a given set of amplification target circles. At least one of the detection rolling circle replication primers can correspond to an amplification target circle in each of at least two of the sets of amplification target circles. Other combinations of sets and relationships of primers to sets and members of the sets are contemplated.

[0136] At least one of the detection rolling circle replication primers corresponding to an amplification target circle in one of the sets of amplification target circles can be labeled or detected with the same fluorescent moiety as at least one of the detection rolling circle replication primers corresponding to an amplification target circle in a different one of the sets of amplification target circles. At least one of the detection rolling circle replication primers corresponding to an amplification target circle in one of the sets of amplification target circles can be labeled or detected with the same fluorescent moiety as at least one of the detection rolling circle replication primers in the same set of amplification target circles.

[0137] Another form of amplification target circle set is a matched amplification target circle set. In one form of matched amplification target circle set, the amplification target circles are circularized open circle probes from a matched set of open circle probes. In a matched open circle probe set, the open circle probes can be targeted to different forms of the same target sequence. Matched open circle probe sets are described further elsewhere herein. In another form of matched amplification target circles, the amplification target circles correspond to or are derived from different forms of the same target molecule. Different forms of a target molecule refer to the same molecule that has some difference. For example, different forms of the same target molecule can include a “wild type” or “normal” form and one or more “mutant” forms of, for example, the same nucleic protein; two or more polymorphic forms of, for example, the same protein. These are only examples. The different forms of the same target molecule can be any set of target molecules that have an overall similarity but that are not identical. By targeting two or more different forms of a target molecule that may be present, the amplification reaction can include a positive control. That is, for example, an amplification target circle corresponding to the normal form of the target molecule will produce a signal even if the mutant form of the target molecule is not present in the reaction or an amplification target circle corresponding to the mutant form of the target molecule will produce a signal even if the normal form of the target molecule is not present in the reaction.

[0138] The set of amplification target circles can include one or a plurality of matched amplification target circle sets. Amplification target circles in different matched amplification target circle sets can correspond to the same or to different target molecules. Thus, open circle probes in one matched amplification target circle set can correspond to different forms of the same target molecule (for example, a first protein) while amplification target circles in a different matched amplification target circle set can correspond to different forms of a different target molecule (for example, a second protein). The different target molecules can be unrelated or can have some relationship to each other. For example, the different target molecules to which amplification target circles in different matched amplification target circle sets correspond can be associated with the same disease or condition. This is useful, for example, for simultaneously testing for the presence of multiple different proteins associated with a disease or condition.

[0139] Matched amplification target circle sets can be subsets of other amplification target circle sets (such as amplification target circle sets having the relationships to primers described above). A set of amplification target circles can comprise one or a plurality of matched amplification target circle sets. Differential detection of different amplification target circles in sets of amplification target circles and in sets of matched amplification target circles can be accomplished generally as described elsewhere herein. For example, different detection rolling circle replication primers can correspond to different amplification target circles in the set and can include or be associated with different fluorescent moieties.

[0140] D. Rolling Circle Replication Primers

[0141] A rolling circle replication primer (RCRP) is an oligonucleotide having sequence complementary to the primer complement portion of an OCP or ATC. This sequence is referred to as the complementary portion of the RCRP. The complementary portion of a RCRP and the cognate primer complement portion can have any desired sequence so long as they are complementary to each other. In general, the sequence of the RCRP can be chosen such that it is not significantly complementary to any other portion of the OCP or ATC. The complementary portion of a rolling circle replication primer can be any length that supports specific and stable hybridization between the primer and the primer complement portion. Generally this is 10 to 35 nucleotides long, but is preferably 16 to 20 nucleotides long. Useful rolling circle replication primers are fluorescent change primers.

[0142] It is preferred that rolling circle replication primers also contain additional sequence at the 5′ end of the RCRP that is not complementary to any part of the OCP or ATC. This sequence is referred to as the non-complementary portion of the RCRP. The non-complementary portion of the RCRP, if present, can serve to facilitate strand displacement during DNA replication. The non-complementary portion of a RCRP may be any length, but is generally 1 to 100 nucleotides long, and preferably 4 to 8 nucleotides long. The non-complementary portion can be involved in interactions that provide specialized effects. For example, the non-complementary portion can comprise a quencher complement portion that can hybridize to a peptide nucleic acid quencher or peptide nucleic acid fluor or that can form an intramolecular structure. Rolling circle replication primers can also comprise fluorescent moieties or labels and quenching moieties.

[0143] Useful rolling circle replication primers for use in the disclosed method can form an intramolecular stem structure involving one or both of the RCRP's ends. Such rolling circle replication primers are referred to herein as hairpin rolling circle replication primers. An intramolecular stem structure involving an end refers to a stem structure where the terminal nucleotides (that is, nucleotides at the end) of the RCRP are hybridized to other nucleotides in the RCRP.

[0144] The intramolecular stem structure can form a hairpin structure or a stem and loop structure. If both ends of an RCRP are involved in an intramolecular stem structure, the two ends of the RCRP can each form a separate intramolecular stem structure or can together form a single intramolecular stem structure. In the latter case the two ends would be hybridized together. It is preferred that the 3′ end of the rolling circle replication primer form an intramolecular stem structure. The 5′ end of the rolling circle replication primer can also form an intramolecular stem structure, either alone, or in the rolling circle replication primer having an intramolecular stem structure at the 3′ end. The intramolecular stem structure preferably involves both ends of the primer and has a blunt end. Also preferred is a short 3′ unpaired overhang. The intramolecular stem structure preferably forms under conditions suitable for nucleic acid replication, and in particular under conditions used for nucleic acid replication when the rolling circle replication primer is being used. For example, the intramolecular stem stricture can be designed to form under conditions used for rolling circle replication. The formation of the intramolecular stem structure during replication allows the structure to reduce or prevent priming by rolling circle replication primers at unintended sequences. In particular, the intramolecular stem structure prevents the rolling circle replication primer in which the structure forms from priming rolling circle replication, from priming nucleic acid replication, or both, at sites other than primer complement sequences (that is, the specific sequences complementary to the complementary portion of the rolling circle replication primer). This follows from the sequestration of the end of rolling circle replication primer in the stem. The end of the rolling circle replication primer cannot hybridize to, and prime from, another sequence while sequestered in the intramolecular stem structure. For this purpose, it is preferred that the intramolecular stem structure be less stable that the hybrid between the primer complement sequence and the complementary portion of the rolling circle replication primer (or, put another way, the hybrid between the primer complement sequence and the complementary portion of the rolling circle replication primer should be more stable than the intramolecular stem structure). It is also preferred that the intramolecular stem structure be more stable than hybrids between the rolling circle replication primer and mismatched sequences. In this way, the intramolecular stem structure will be thermodynamically favored over undesired primer hybridizations. Although rolling circle replication primers that form intramolecular stem structures at the 3′ end leaving the 5′ end unpaired and overhanging can be used, this is not preferred. In such a case, the 3′ end could be extended during replication (using rolling circle replication primer sequences as template), thus inactivating the primers.

[0145] Where the intramolecular stem structure of a rolling circle replication primer forms a stem and loop structure, it is preferred that a portion of the complementary portion of the rolling circle replication primer be in the loop of the stem and loop structure. This portion of the complementary portion in the loop can then hybridize to the primer complement sequence of the open circle probe. Such an arrangement allows design of hairpin rolling circle replication primers where the stability of the intramolecular stem structure depends on the presence or absence of the specific primer complement sequence. In particular, a rolling circle replication primer that forms a stem and loop structure with a portion of the complementary portion in the loop can be designed so that hybridization of the complementary portion in the loop to the primer complement sequence disrupts the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (11999)). In this way, the intramolecular stem structure remains intact in the absence of the primer complement sequence and thus reduces or eliminates the ability of the rolling circle replication primer to prime nucleic acid replication. In the presence of the primer complement sequence, disruption of the intramolecular stem structure allows the end of the rolling circle replication primer to hybridize to the primer complement sequence. This hybrid between the primer complement sequence and the end of the rolling circle replication primer allows the priming of nucleic acid replication by the primer. For this form of hairpin rolling circle replication primer, it is preferred that hybridization of the loop to a sequence other than the primer complement sequence does not disrupt the intramolecular stem structure. Preferably, the hybrid between the primer complement sequence and the end of the rolling circle replication primer is more stable than the intramolecular stem structure. This helps stabilize hybridization of the rolling circle replication primer to the primer complement sequence in competition with the intramolecular stem structure.

[0146] Discrimination of rolling circle replication primer hybridization also can be accomplished by hybridizing primer to primer complement portions of OCPs or ATCs under conditions that favor only exact sequence matches leaving other rolling circle replication primer unhybridized. The unhybridized rolling circle replication primers will retain or re-form the intramolecular hybrid.

[0147] The rolling circle replication primer may also include modified nucleotides to make it resistant to exonuclease digestion. For example, the primer can have three or four phosphorothioate linkages between nucleotides at the 5′ end of the primer. Such nuclease resistant primers allow selective degradation of excess unligated OCP and gap oligonucleotides that might otherwise interfere with hybridization of detection probes, address probes, and secondary OCPs to the amplified nucleic acid. A rolling circle replication primer can be used as the tertiary DNA strand displacement primer in strand displacement cascade amplification.

[0148] Rolling circle replication primers may also include modified nucleotides to make them resistant to exonuclease digestion. For example, the primer can have three or four phosphorothioate linkages between nucleotides at the 5′ end of the primer. Such nuclease resistant primers allow selective degradation of excess unligated OCP and gap oligonucleotides that might otherwise interfere with hybridization of detection probes, address probes, and secondary OCPs to the amplified nucleic acid.

[0149] A rolling circle replication primer is specific for, or corresponds to, an open circle probe or amplification target circle when the complementary portion of the rolling circle replication primer is complementary to the primer complement portion of the open circle probe or amplification target circle. A rolling circle replication primer is not specific for, or does not correspond to, an open circle probe or amplification target circle when the complementary portion of the rolling circle replication primer is not substantially complementary to the open circle probe or amplification target circle. A complementary portion is not substantially complementary to another sequence if it has a melting temperature 10° C. lower than the melting temperature under the same conditions of a sequence fully complementary to the complementary portion of the rolling circle replication primer.

[0150] A rolling circle replication primer is specific for, or corresponds to, a set of open circle probes or a set of amplification target circles when the complementary portion of the rolling circle replication primer is complementary to the primer complement portion of the open circle probes or amplification target circles in the set. A rolling circle replication primer is not specific for, or does not correspond to, a set of open circle probes or a set of amplification target circles when the complementary portion of the rolling circle replication primer is not substantially complementary to the open circle probes or amplification target circles in the set.

[0151] 1. Detection Rolling Circle Replication Primers

[0152] Detection rolling circle replication primers are rolling circle replication primers that are specific for, or correspond to, a particular open circle probe, amplification target circle, set of open circle probes, or set of amplification target circles in an amplification reaction. Useful detection rolling circle replication primers are fluorescent change primers. Fluorescent change primers are primers that involve a change in fluorescence intensity or wavelength based on a change in the form or conformation of the primer. Useful fluorescent change primers have a stem structure with the fluorescent moiety and quenching moiety incorporated into opposite strands of the stem structure. The stem structure can be an intermolecular stem structure (such as a hairpin or stem and loop) or an intramolecular stem structure. In the structured state, the quenching moiety prevents or limits fluorescence of the fluorescent moiety. When the stem of the primer is disrupted, the quenching moiety and fluorescent moiety are no longer in proximity and the fluorescent moiety produces a fluorescent signal. Fluorescent change primers that form an intramolecular stem structure are referred to as hairpin quenched primers. Fluorescent change primers that form an intermolecular stem structure are referred to as stem quenched primers.

[0153] In the disclosed method, use of fluorescent change primers produces double-stranded tandem sequence DNA where the primer stem is disrupted in favor of a complementary, replicated strand. From a reaction initially containing structured (that is, non-fluorescent) fluorescent change primers, fluorescence signal increases as amplification takes place, as more and more of the fluorescent change primers are incorporated into double stranded TS-DNA, as the fluorescent change primer stems are disrupted, and as the fluorescent moieties are consequently unquenched. Thus, use of fluorescent change primers is particularly suited for real-time detection of amplification in ERCA. Examples of fluorescent change primers are stem quenched primers, hairpin quenched primers, Amplifluor primers and scorpion primers.

[0154] 2. Common Rolling Circle Replication Primers

[0155] Common rolling circle replication primers are rolling circle replication primers that are specific for, or correspond to, all of the open circle probes or amplification target circles in an amplification reaction or in a set of open circle probes or set of amplification target circles in an amplification reaction. Common rolling circle replication primers can be fluorescent change primers although this is not preferred.

[0156] The use of both detection rolling circle replication primers and common rolling circle replication primers in a reaction can increase the consistency of the amplification. Detection rolling circle replication primers and common rolling circle replication primers can have different relationships to open circle probes and amplification target circles used in the method. For example, for a given set of open circle probes or amplification target circles, detection rolling circle replication primers can each correspond to a different open circle probe or amplification target circle in the set while common rolling circle replication primers can correspond to all of the open circle probes or amplification target circles in the set. These relationships allow the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of one of the primers used (and its complement on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency). Differential detection can be mediated by the circle-specific detection rolling circle replication primers. The use of secondary DNA strand displacement primers that correspond to all of the open circle probes or amplification target circles in the set has a similar effect of allowing the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of two of the primers used (and their complements on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency).

[0157] The use of two or more rolling circle replication primers, such as the use of a detection rolling circle replication primer and a common rolling circle replication primer, with each primer specific for a different sequence or region of the open circle probes or amplification target circles, can increase the efficiency of amplification by producing multiple simultaneous initiations of replication. For example, each of two or more different rolling circle replication primers can simultaneously prime replication from different sequences in a given circularized open circle probe or amplification target circle. This multiplies the yield of amplification.

[0158] E. DNA Strand Displacement Primers

[0159] Primers used for secondary DNA strand displacement are referred to herein as DNA strand displacement primers. One form of DNA strand displacement primer, referred to herein as a secondary DNA strand displacement primer, is an oligonucleotide having sequence matching part of the sequence of an OCP or ATC. This sequence in the secondary DNA strand displacement primer is referred to as the matching portion of the secondary DNA strand displacement primer. The sequence in the OCP or ATC that matches the matching portion of the secondary DNA strand displacement primer is referred to as the secondary DNA strand displacement primer matching portion. The matching portion of a secondary DNA strand displacement primer is complementary to sequences in TS-DNA. The matching portion of a secondary DNA strand displacement primer may be complementary to any sequence in TS-DNA. However, it is preferred that it not be complementary TS-DNA sequence matching either the rolling circle replication primers or a tertiary DNA strand displacement primer, if one is being used. This prevents hybridization of the primers to each other. The matching portion of a secondary DNA strand displacement primer may be complementary to all or a portion of the target sequence. In this case, it is preferred that the 3′ end nucleotides of the secondary DNA strand displacement primer are complementary to the gap sequence in the target sequence. It is most preferred that nucleotide at the 3′ end of the secondary DNA strand displacement primer falls complementary to the last nucleotide in the gap sequence of the target sequence, that is, the 5′ nucleotide in the gap sequence of the target sequence. The matching portion of a secondary DNA strand displacement primer can be any length that supports specific and stable hybridization between the primer and its complement. Generally this is 12 to 35 nucleotides long, but is preferably 18 to 25 nucleotides long.

[0160] Secondary DNA strand displacement primers can be specific for, or correspond to, all of the open circle probes or amplification target circles in an amplification reaction or in a set of open circle probes or set of amplification target circles in an amplification reaction. A secondary DNA strand displacement primer is specific for, or corresponds to, an open circle probe or amplification target circle when the matching portion of the secondary DNA strand displacement primer matches the primer complement portion of the open circle probe or amplification target circle. A secondary DNA strand displacement primer is not specific for, or does not correspond to, an open circle probe or amplification target circle when the matching portion of the secondary DNA strand displacement primer does not substantially match sequence in the open circle probe or amplification target circle. A matching portion does not substantially match another sequence if it has a melting temperature with the complement of the other sequence that is 10° C. lower than the melting temperature under the same conditions of a sequence fully complementary to the matching portion of the secondary DNA strand displacement primer.

[0161] A secondary DNA strand displacement primer is specific for, or corresponds to, a set of open circle probes or a set of amplification target circles when the matching portion of the secondary DNA strand displacement primer matches the primer complement portion of the open circle probes or amplification target circles in the set. A secondary DNA strand displacement primer is not specific for, or does not correspond to, a set of open circle probes or a set of amplification target circles when the matching portion of the secondary DNA strand displacement primer does not substantially match the open circle probes or amplification target circles in the set. Secondary DNA strand displacement primers can be fluorescent change primers although this is not preferred.

[0162] The use of two or more rolling circle replication primers, such as the use of a detection rolling circle replication primer and a common rolling circle replication primer, with each primer specific for a different sequence or region of the open circle probes or amplification target circles, can increase the efficiency of amplification by producing multiple simultaneous generations of amplification product. For example, use of both rolling circle replication primers (which prime replication of circularized open circle probes and amplification target circles) and secondary DNA strand displacement primers (which prime replication of the product of replication of circularized open circle probes and amplification target circles) allows multiple generations of amplification product to be generated simultaneously. This multiplies the yield of amplification.

[0163] The use of both rolling circle replication primers and secondary DNA strand displacement primers in a reaction can increase the consistency of the amplification. rolling circle replication primers and secondary DNA strand displacement primers can have different relationships to open circle probes and amplification target circles used in the method. For example, for a given set of open circle probes or amplification target circles, detection rolling circle replication primers can each correspond to a different open circle probe or amplification target circle in the set while secondary DNA strand displacement primers and common rolling circle replication primers can correspond to all of the open circle probe or amplification target circles in the set. These relationships allow the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of two of the primers used (and their complements on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency). Differential detection is mediated by the circle-specific detection rolling circle replication primers.

[0164] It is preferred that secondary DNA strand displacement primers also contain additional sequence at the 5′ end of the primer that does not match any part of the OCP or ATC. This sequence is referred to as the non-matching portion of the secondary DNA strand displacement primer. The non-matching portion of the secondary DNA strand displacement primer, if present, can serve to facilitate strand displacement during DNA replication. The non-matching portion of a secondary DNA strand displacement primer may be any length, but is generally 1 to 100 nucleotides long, and preferably 4 to 8 nucleotides long. The non-matching portion can be involved in interactions that provide specialized effects. For example, the non-matching portion can comprise a quencher complement portion that can hybridize to a peptide nucleic acid quencher or peptide nucleic acid fluor or that can form an intramolecular structure. Secondary DNA strand displacement primers can also comprise fluorescent moieties or labels and quenching moieties.

[0165] Useful secondary DNA strand displacement primers for use in the disclosed method can form an intramolecular stem structure involving one or both of the secondary DNA strand displacement primer's ends. Such secondary DNA strand displacement primers are referred to herein as hairpin secondary DNA strand displacement primers. An intramolecular stem structure involving an end refers to a stem structure where the terminal nucleotides (that is, nucleotides at the end) of the secondary DNA strand displacement primer are hybridized to other nucleotides in the secondary DNA strand displacement primer.

[0166] The intramolecular stem structure can form a hairpin structure or a stem and loop structure. If both ends of a secondary DNA strand displacement primer are involved in an intramolecular stem structure, the two ends of the secondary DNA strand displacement primer can each form a separate intramolecular stem structure or can together form a single intramolecular stem structure. In the latter case the two ends would be hybridized together. It is preferred that the 3′ end of the secondary DNA strand displacement primer form an intramolecular stem structure. The 5′ end of the secondary DNA strand displacement primer can also form an intramolecular stem structure, either alone, or in the secondary DNA strand displacement primer having an intramolecular stem structure at the 3′ end. The intramolecular stem structure preferably involves both ends of the primer and has a blunt end. Also preferred is a short 3′ unpaired overhang. The intramolecular stem structure preferably forms under conditions suitable for nucleic acid replication, and in particular under conditions used for nucleic acid replication when the secondary DNA strand displacement primer is being used.

[0167] For example, the intramolecular stem structure can be designed to form under conditions used for rolling circle replication. The formation of the intramolecular stem structure during replication allows the structure to reduce or prevent priming by secondary DNA strand displacement primers at unintended sequences. In particular, the intramolecular stem structure prevents the secondary DNA strand displacement primer in which the structure forms from priming nucleic acid replication at sites other than primer complement sequences (that is, the specific sequences complementary to the complementary portion of the secondary DNA strand displacement primer) in TS-DNA. This follows from the sequestration of the end of secondary DNA strand displacement primer in the stem. The end of the rolling circle replication primer cannot hybridize to, and prime from, another sequence while sequestered in the intramolecular stem structure. For this purpose, it is preferred that the intramolecular stem structure be less stable that the hybrid between the primer complement sequence and the complementary portion of the secondary DNA strand displacement primer (or, put another way, the hybrid between the primer complement sequence and the matching portion of the secondary DNA strand displacement primer should be more stable than the intramolecular stem structure). It is also preferred that the intramolecular stem structure be more stable than hybrids between the secondary DNA strand displacement primer and mismatched sequences. In this way, the intramolecular stem structure will be thermodynamically favored over undesired primer hybridizations. Although secondary DNA strand displacement primers that form intramolecular stem structures at the 3′ end leaving the 5′ end unpaired and overhanging can be used, they are not preferred. In such a case, the 3′ end could be extended during replication (using secondary DNA strand displacement primer sequences as template), thus inactivating the primers.

[0168] Where the intramolecular stem structure of a secondary DNA strand displacement primer forms a stem and loop structure, it is preferred that a portion of the complementary portion of the secondary DNA strand displacement primer be in the loop of the stem and loop structure. This portion of the complementary portion in the loop can then hybridize to the primer complement sequence in TS-DNA. Such an arrangement allows design of hairpin secondary DNA strand displacement primers where the stability of the intramolecular stem structure depends on the presence or absence of the specific primer complement sequence. In particular, a secondary DNA strand displacement primer that forms a stem and loop structure with a portion of the matching portion in the loop can be designed so that hybridization of the matching portion in the loop to the primer complement sequence disrupts the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). In this way, the intramolecular stem structure remains intact in the absence of the primer complement sequence and thus reduces or eliminates the ability of the secondary DNA strand displacement primer to prime nucleic acid replication. In the presence of the primer complement sequence, disruption of the intramolecular stem structure allows the end of the secondary DNA strand displacement primer to hybridize to the primer complement sequence. This hybrid between the primer complement sequence and the end of the secondary DNA strand displacement primer allows the priming of nucleic acid replication by the primer. For this form of hairpin secondary DNA strand displacement primer, it is preferred that hybridization of the loop to a sequence other than the primer complement sequence does not disrupt the intramolecular stem structure. Preferably, the hybrid between the primer complement sequence and the end of the secondary DNA strand displacement primer is more stable than the intramolecular stem structure. This helps stabilize hybridization of the secondary DNA strand displacement primer to the primer complement sequence in competition with the intramolecular stem structure.

[0169] Discrimination of secondary DNA strand displacement primer hybridization also can be accomplished by hybridizing primer to primer complement portions in TS-DNA under conditions that favor only exact sequence matches leaving other secondary DNA strand displacement primer unhybridized. The unhybridized secondary DNA strand displacement primers will retain or re-form the intramolecular hybrid.

[0170] Another form of DNA strand displacement primer, referred to herein as a tertiary DNA strand displacement primer, is an oligonucleotide having sequence complementary to part of the sequence of an OCP or ATC. This sequence is referred to as the complementary portion of the tertiary DNA strand displacement primer. This complementary portion of the tertiary DNA strand displacement primer matches sequences in TS-DNA. The complementary portion of a tertiary DNA strand displacement primer may be complementary to any sequence in the OCP or ATC. However, it is preferred that it not be complementary OCP or ATC sequence matching the secondary DNA strand displacement primer. This prevents hybridization of the primers to each other. Preferably, the complementary portion of the tertiary DNA strand displacement primer has sequence complementary to a portion of the spacer portion of an OCP. The complementary portion of a tertiary DNA strand displacement primer can be any length that supports specific and stable hybridization between the primer and its complement. Generally this is 12 to 35 nucleotides long, but is preferably 18 to 25 nucleotides long. Tertiary DNA strand displacement primers can be fluorescent change primers although this is not preferred.

[0171] Useful tertiary DNA strand displacement primers for use in the disclosed method can form an intramolecular stem structure involving one or both of the tertiary DNA strand displacement primer's ends. Such tertiary DNA strand displacement primers are referred to herein as hairpin tertiary DNA strand displacement primers. An intramolecular stem structure involving an end refers to a stem structure where the terminal nucleotides (that is, nucleotides at the end) of the tertiary DNA strand displacement primer are hybridized to other nucleotides in the tertiary DNA strand displacement primer.

[0172] The intramolecular stem structure can form a hairpin structure or a stem and loop structure. If both ends of a tertiary DNA strand displacement primer are involved in an intramolecular stem structure, the two ends of the tertiary DNA strand displacement primer can each form a separate intramolecular stem structure or can together form a single intramolecular stem structure. In the latter case the two ends would be hybridized together. It is preferred that the 3′ end of the tertiary DNA strand displacement primer form an intramolecular stem structure. The 5′ end of the tertiary DNA strand displacement primer can also form an intramolecular stem structure, either alone, or in the tertiary DNA strand displacement primer having an intramolecular stem structure at the 3′ end. The intramolecular stem structure preferably forms under conditions suitable for nucleic acid replication, and in particular under conditions used for nucleic acid replication when the tertiary DNA strand displacement primer is being used. For example, the intramolecular stem structure can be designed to form under conditions used for rolling circle replication. The formation of the intramolecular stem structure during replication allows the structure to reduce or prevent priming by tertiary DNA strand displacement primers at unintended sequences. In particular, the intramolecular stem structure prevents the tertiary DNA strand displacement primer in which the structure forms from priming nucleic acid replication at sites other than primer complement sequences (that is, the specific sequences complementary to the complementary portion of the tertiary DNA strand displacement primer) in TS-DNA. This follows from the sequestration of the end of tertiary DNA strand displacement primer in the stem. The end of the rolling circle replication primer cannot hybridize to, and prime from, another sequence while sequestered in the intramolecular stem structure. For this purpose, it is preferred that the intramolecular stem structure be less stable that the hybrid between the primer complement sequence and the complementary portion of the tertiary DNA strand displacement primer (or, put another way, the hybrid between the primer complement sequence and the complementary portion of the tertiary DNA strand displacement primer should be more stable than the intramolecular stem structure). It is also preferred that the intramolecular stem structure be more stable than hybrids between the tertiary DNA strand displacement primer and mismatched sequences. In this way, the intramolecular stem structure will be thermodynamically favored over undesired primer hybridizations. Tertiary DNA strand displacement primers that form intramolecular stem structures at the 3′ end will have the 3′ end extended during replication (using tertiary DNA strand displacement primer sequences as template). This serves to stabilize the intramolecular stem structure in the tertiary DNA strand displacement primers, making them unavailable for priming.

[0173] Where the intramolecular stem structure of a tertiary DNA strand displacement primer forms a stem and loop structure, it is preferred that a portion of the complementary portion of the tertiary DNA strand displacement primer be in the loop of the stem and loop structure. This portion of the complementary portion in the loop can then hybridize to the primer complement sequence in TS-DNA. Such an arrangement allows design of hairpin tertiary DNA strand displacement primers where the stability of the intramolecular stem structure depends on the presence or absence of the specific primer complement sequence. In particular, a tertiary DNA strand displacement primer that forms a stem and loop structure with a portion of the complementary portion in the loop can be designed so that hybridization of the complementary portion in the loop to the primer complement sequence disrupts the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). In this way, the intramolecular stem structure remains intact in the absence of the primer complement sequence and thus reduces or eliminates the ability of the tertiary DNA strand displacement primer to prime nucleic acid replication. In the presence of the primer complement sequence, disruption of the intramolecular stem structure allows the end of the tertiary DNA strand displacement primer to hybridize to the primer complement sequence. This hybrid between the primer complement sequence and the end of the tertiary DNA strand displacement primer allows the priming of nucleic acid replication by the primer. For this form of hairpin tertiary DNA strand displacement primer, it is preferred that hybridization of the loop to a sequence other than the primer complement sequence does not disrupt the intramolecular stem structure. Preferably, the hybrid between the primer complement sequence and the end of the tertiary DNA strand displacement primer is more stable than the intramolecular stem structure. This helps stabilize hybridization of the tertiary DNA strand displacement primer to the primer complement sequence in competition with the intramolecular stem structure.

[0174] Discrimination of tertiary DNA strand displacement primer hybridization also can be accomplished by hybridizing primer to primer complement portions in TS-DNA under conditions that favor only exact sequence matches leaving other tertiary DNA strand displacement primer unhybridized. The unhybridized tertiary DNA strand displacement primers will retain or re-form the intramolecular hybrid and the end of the tertiary DNA strand displacement primer involved in the intramolecular stem structure will be extended during replication.

[0175] It is preferred that tertiary DNA strand displacement primers also contain additional sequence at their 5′ end that is not complementary to any part of the OCP or ATC. This sequence is referred to as the non-complementary portion of the tertiary DNA strand displacement primer. The non-complementary portion of the tertiary DNA strand displacement primer, if present, serves to facilitate strand displacement during DNA replication. The non-complementary portion of a tertiary DNA strand displacement primer may be any length, but is generally 1 to 100 nucleotides long, and preferably 4 to 8 nucleotides long. A rolling circle replication primer is a preferred form of tertiary DNA strand displacement primer. Secondary DNA strand displacement primers can also comprise fluorescent moieties or labels and quenching moieties.

[0176] DNA strand displacement primers may also include modified nucleotides to make them resistant to exonuclease digestion. For example, the primer can have three or four phosphorothioate linkages between nucleotides at the 5′ end of the primer. Such nuclease resistant primers allow selective degradation of excess unligated OCP and gap oligonucleotides that might otherwise interfere with hybridization of detection probes, address probes, and secondary OCPs to the amplified nucleic acid. DNA strand displacement primers can be used for secondary DNA strand displacement and strand displacement cascade amplification, both described below and in U.S. Pat. No. 6,143,495.

[0177] F. Fluorescent Change Probes and Primers

[0178] Fluorescent change probes and fluorescent change primers refer to all probes and primers that involve a change in fluorescence intensity or wavelength based on a change in the form of conformation of the probe or primer and nucleic acid to be detected, assayed or replicated. Examples of fluorescent change probes and primers include molecular beacons, Amplifluors, FRET probes, cleavable FRET probes, TaqMan probes, scorpion primers, fluorescent triplex oligos, fluorescent water-soluble conjugated polymers, PNA probes and QPNA probes.

[0179] Fluorescent change probes and primers can be classified according to their structure and/or function. Fluorescent change probes include hairpin quenched probes, cleavage quenched probes, cleavage activated probes, and fluorescent activated probes.

[0180] Fluorescent change primers include stem quenched primers and hairpin quenched primers. The use of several types of fluorescent change probes and primers are reviewed in Schweitzer and Kingsmore, Curr. Opin. Biotech. 12:21-27 (2001). Hall et al., Proc. Natl. Acad. Sci. USA 97:8272-8277 (2000), describe the use of fluorescent change probes with Invader assays.

[0181] Hairpin quenched probes are probes that when not bound to a target sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the probe binds to a target sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Examples of hairpin quenched probes are molecular beacons, fluorescent triplex oligos, and QPNA probes.

[0182] Cleavage activated probes are probes where fluorescence is increased by cleavage of the probe. Cleavage activated probes can include a fluorescent label and a quenching moiety in proximity such that fluorescence from the label is quenched. When the probe is clipped or digested (typically by the 5′-3′ exonuclease activity of a polymerase during amplification), the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. TaqMan probes (Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280 (1991)) are an example of cleavage activated probes.

[0183] Cleavage quenched probes are probes where fluorescence is decreased or altered by cleavage of the probe. Cleavage quenched probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity, fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. The probes are thus fluorescent, for example, when hybridized to a target sequence. When the probe is clipped or digested (typically by the 5′-3′ exonuclease activity of a polymerase during amplification), the donor moiety is no longer in proximity to the acceptor fluorescent label and fluorescence from the acceptor decreases. If the donor moiety is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor. The overall effect would then be a reduction of acceptor fluorescence and an increase in donor fluorescence. Donor fluorescence in the case of cleavage quenched probes is equivalent to fluorescence generated by cleavage activated probes with the acceptor being the quenching moiety and the donor being the fluorescent label. Cleavable FRET (fluorescence resonance energy transfer) probes are an example of cleavage quenched probes.

[0184] Fluorescent activated probes are probes or pairs of probes where fluorescence is increased or altered by hybridization of the probe to a target sequence. Fluorescent activated probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity (when the probes are hybridized to a target sequence), fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. Fluorescent activated probes are typically pairs of probes designed to hybridize to adjacent sequences such that the acceptor and donor are brought into proximity. Fluorescent activated probes can also be single probes containing both a donor and acceptor where, when the probe is not hybridized to a target sequence, the donor and acceptor are not in proximity but where the donor and acceptor are brought into proximity when the probe hybridized to a target sequence. This can be accomplished, for example, by placing the donor and acceptor on opposite ends a the probe and placing target complement sequences at each end of the probe where the target complement sequences are complementary to adjacent sequences in a target sequence. If the donor moiety of a fluorescent activated probe is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor (that is, when the probes are not hybridized to the target sequence). When the probes hybridize to a target sequence, the overall effect would then be a reduction of donor fluorescence and an increase in acceptor fluorescence. FRET probes are an example of fluorescent activated probes.

[0185] Stem quenched primers are primers that when not hybridized to a complementary sequence form a stem structure (either an intramolecular stem structure or an intermolecular stem structure) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. In the disclosed method, stem quenched primers are used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of stem quenched primers are peptide nucleic acid quenched primers and hairpin quenched primers.

[0186] Peptide nucleic acid quenched primers are primers associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor to form a stem structure. The primer contains a fluorescent label or a quenching moiety and is associated with either a peptide nucleic acid quencher or a peptide nucleic acid fluor, respectively. This puts the fluorescent label in proximity to the quenching moiety. When the primer is replicated, the peptide nucleic acid is displaced, thus allowing the fluorescent label to produce a fluorescent signal.

[0187] Hairpin quenched primers are primers that when not hybridized to a complementary sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Hairpin quenched primers are typically used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of hairpin quenched primers are Amplifluor primers (Nazerenko et al., Nucleic Acids Res. 25:2516-2521 (1997)) and scorpion primers (Thelwell et al., Nucleic Acids Res. 28(19):3752-3761 (2000)).

[0188] Cleavage activated primers are similar to cleavage activated probes except that they are primers that are incorporated into replicated strands and are then subsequently cleaved. Little et al., Clin. Chem. 45:777-784 (1999), describe the use of cleavage activated primers.

[0189] G. Reporter Binding Agents

[0190] A reporter binding agent is a specific binding molecule coupled or tethered to a nucleic acid such as an oligonucleotide. The specific binding molecule is referred to as the affinity portion of the reporter binding agent and the nucleic acid is referred to as the oligonucleotide portion of the reporter binding agent. As used herein, a specific binding molecule is a molecule that interacts specifically with a particular molecule or moiety (that is, an analyte). The molecule or moiety that interacts specifically with a specific binding molecule is referred to herein as a target molecule. The target molecules can be any analyte. It is to be understood that the term target molecule refers to both separate molecules and to portions of molecules, such as an epitope of a protein, that interacts specifically with a specific binding molecule. Antibodies, either member of a receptor/ligand pair, and other molecules with specific binding affinities are examples of specific binding molecules, useful as the affinity portion of a reporter binding molecule. A reporter binding molecule with an affinity portion which is an antibody is referred to herein as a reporter antibody. The oligonucleotide portion can be a nucleic acid molecule or a combination of nucleic acid molecules. The oligonucleotide portion is preferably an oligonucleotide or an amplification target circle.

[0191] By tethering an amplification target circle or coupling a target sequence to a specific binding molecule, binding of a specific binding molecule to its specific target can be detected by amplifying the ATC or target sequence with rolling circle amplification. This amplification allows sensitive detection of a very small number of bound specific binding molecules. A reporter binding molecule that interacts specifically with a particular target molecule is said to be specific for that target molecule. For example, a reporter binding molecule with an affinity portion which is an antibody that binds to a particular antigen is said to be specific for that antigen. The antigen is the target molecule. Reporter binding agents are also referred to herein as reporter binding molecules. FIGS. 25, 26, 27, 28, and 29 of U.S. Pat. No. 6,143,495 illustrate examples of several preferred types of reporter binding molecules and their use. FIG. 29 of U.S. Pat. No. 6,143,495 illustrates a reporter binding molecule using an antibody as the affinity portion.

[0192] Preferred target molecules are proteins and peptides. Use of reporter binding agents that target proteins and peptides allows sensitive signal amplification using rolling circle amplification for the detection of proteins and peptides. The ability to multiplex rolling circle amplification detection allows multiplex detection of the proteins and peptides (or any other target molecule). Thus, the disclosed method can be used for multi-protein analysis such as proteomics analysis. Such multi-protein analysis can be accomplished, for example, by using reporter binding agents targeted to different proteins, with the oligonucleotide portion of each reporter binding agent coded to allow separate amplification and detection of each different reporter binding agent.

[0193] In one embodiment, the oligonucleotide portion of a reporter binding agent includes a sequence, referred to as a target sequence, that serves as a target sequence for an OCP. The sequence of the target sequence can be arbitrarily chosen. In a multiplex assay using multiple reporter binding agents, it is preferred that the target sequence for each reporter binding agent be substantially different to limit the possibility of non-specific target detection. Alternatively, it may be desirable in some multiplex assays, to use target sequences with related sequences. By using different, unique gap oligonucleotides to fill different gap spaces, such assays can use one or a few OCPs to amplify and detect a larger number of target sequences. The oligonucleotide portion can be coupled to the affinity portion by any of several established coupling reactions. For example, Hendrickson et al., Nucleic Acids Res., 23(3):522-529 (1995) describes a suitable method for coupling oligonucleotides to antibodies.

[0194] A preferred form of target sequence in a reporter binding agent is an oligonucleotide having both ends coupled to the specific binding molecule so as to form a loop. In this way, when the OCP hybridizes to the target and is circularized, the OCP will remain topologically locked to the reporter binding agent during rolling circle replication of the circularized OCP. This improves the localization of the resulting amplified signal to the location where the reporter binding agent is bound (that is, at the location of the target molecule).

[0195] A special form of reporter binding molecule, referred to herein as a reporter binding probe, has an oligonucleotide or oligonucleotide derivative as the specific binding molecule. Reporter binding probes are designed for and used to detect specific nucleic acid sequences. Thus, the target molecule for reporter binding probes are nucleic acid sequences. The target molecule for a reporter binding probe can be a nucleotide sequence within a larger nucleic acid molecule. It is to be understood that the term reporter binding molecule encompasses reporter binding probes. The specific binding molecule of a reporter binding probe can be any length that supports specific and stable hybridization between the reporter binding probe and the target molecule. For this purpose, a length of 10 to 40 nucleotides is preferred, with a specific binding molecule of a reporter binding probe 16 to 25 nucleotides long being most preferred. It is preferred that the specific binding molecule of a reporter binding probe is peptide nucleic acid. As described above, peptide nucleic acid forms a stable hybrid with DNA. This allows a reporter binding probe with a peptide nucleic acid specific binding molecule to remain firmly adhered to the target sequence during subsequent amplification and detection operations. This useful effect can also be obtained with reporter binding probes with oligonucleotide specific binding molecules by making use of the triple helix chemical bonding technology described by Gasparro et al., Nucleic Acids Res. 1994 22(14):2845-2852 (1994). Briefly, the affinity portion of a reporter binding probe is designed to form a triple helix when hybridized to a target sequence. This is accomplished generally as known, preferably by selecting either a primarily homopurine or primarily homopyrimidine target sequence. The matching oligonucleotide sequence which constitutes the affinity portion of the reporter binding probe will be complementary to the selected target sequence and thus be primarily homopyrimidine or primarily homopurine, respectively. The reporter binding probe (corresponding to the triple helix probe described by Gasparro et al.) contains a chemically linked psoralen derivative. Upon hybridization of the reporter binding probe to a target sequence, a triple helix forms. By exposing the triple helix to low wavelength ultraviolet radiation, the psoralen derivative mediates cross-linking of the probe to the target sequence. FIGS. 25, 26, 27, and 28 of U.S. Pat. No. 6,143,495 illustrate examples of reporter binding molecules that are reporter binding probes.

[0196] The specific binding molecule in a reporter binding probe can also be a bipartite DNA molecule, such as ligatable DNA probes adapted from those described by Landegren et al., Science 241:1077-1080 (1988). When using such a probe, the affinity portion of the probe is assembled by target-mediated ligation of two oligonucleotide portions which hybridize to adjacent regions of a target nucleic acid. Thus, the components used to form the affinity portion of such reporter binding probes are a truncated reporter binding probe (with a truncated affinity portion which hybridizes to part of the target sequence) and a ligation probe which hybridizes to an adjacent part of the target sequence such that it can be ligated to the truncated reporter binding probe. The ligation probe can also be separated from (that is, not adjacent to) the truncated reporter binding probe when both are hybridized to the target sequence. The resulting space between them can then be filled by a second ligation probe or by gap-filling synthesis. For use in the disclosed methods, it is preferred that the truncated affinity portion be long enough to allow target-mediated ligation but short enough to, in the absence of ligation to the ligation probe, prevent stable hybridization of the truncated reporter binding probe to the target sequence during the subsequent amplification operation. For this purpose, a specific step designed to eliminate hybrids between the target sequence and unligated truncated reporter binding probes can be used following the ligation operation.

[0197] In another embodiment, the oligonucleotide portion of a reporter binding agent includes a sequence, referred to as a rolling circle replication primer sequence, that serves as a rolling circle replication primer for an ATC. This allows rolling circle replication of an added ATC where the resulting TS-DNA is coupled to the reporter binding agent. Because of this, the TS-DNA will be effectively immobilized at the site of the target molecule. Preferably, the immobilized TS-DNA can then be collapsed in situ prior to detection. The sequence of the rolling circle replication primer sequence can be arbitrarily chosen. The rolling circle replication sequence can be designed to form and intramolecular stem structure as described for rolling circle replication primers above.

[0198] In a multiplex assay using multiple reporter binding agents, it is preferred that the detection rolling circle replication primer sequences for each reporter binding agent be substantially different to limit the possibility of non-specific target detection. Alternatively, it may be desirable in some multiplex assays, to use detection rolling circle replication primer sequences with related sequences. Such assays can use one or a few ATCs to detect a larger number of target molecules. It is preferred that common rolling circle replication primer sequences for each reporter binding agent be the same. Any of the other relationships between ATCs and primers disclosed herein can also be used. When the oligonucleotide portion of a reporter binding agent is used as a rolling circle replication primer, the oligonucleotide portion can be any length that supports specific and stable hybridization between the oligonucleotide portion and the primer complement portion of an amplification target circle. Generally this is 10 to 35 nucleotides long, but is preferably 16 to 20 nucleotides long. FIGS. 25, 26, 27, 28, and 29 of U.S. Pat. No. 6,143,495 illustrate examples of reporter binding molecules in which the oligonucleotide portion is a rolling circle replication primer.

[0199] In another embodiment, the oligonucleotide portion of a reporter binding agent can include an amplification target circle which serves as a template for rolling circle replication. In a multiplex assay using multiple reporter binding agents, it is preferred that detection primer complement portions, address tag portions, and detection tag portions of the ATC comprising the oligonucleotide portion of each reporter binding agent be substantially different to unique detection of each reporter binding agent. It is desirable, however, to use the same common primer complement portion in all of the ATCs used in a multiplex assay. The ATC is tethered to the specific binding molecule by looping the ATC around a tether loop. This allows the ATC to rotate freely during rolling circle replication while remaining coupled to the affinity portion. The tether loop can be any material that can form a loop and be coupled to a specific binding molecule. Linear polymers are a preferred material for tether loops.

[0200] A preferred method of producing a reporter binding agent with a tethered ATC is to form the tether loop by ligating the ends of oligonucleotides coupled to a specific binding molecule around an ATC. Oligonucleotides can be coupled to specific binding molecules using known techniques. For example, Hendrickson et al. (1995), describes a suitable method for coupling oligonucleotides to antibodies. This method is generally useful for coupling oligonucleotides to any protein. To allow ligation, oligonucleotides comprising the two halves of the tether loop should be coupled to the specific binding molecule in opposite orientations such that the free end of one is the 5′ end and the free end of the other is the 3′ end. Ligation of the ends of the tether oligonucleotides can be mediated by hybridization of the ends of the tether oligonucleotides to adjacent sequences in the ATC to be tethered. In this way, the ends of the tether oligonucleotides are analogous to the target probe portions of an open circle probe, with the ATC containing the target sequence. Similar techniques can be used to form tether loops containing a target sequence.

[0201] Another preferred method of producing a reporter binding agent with a tethered ATC is to ligate an open circle probe while hybridized to an oligonucleotide tether loop on a specific binding molecule. In this method, both ends of a single tether oligonucleotide are coupled to a specific binding molecule. This can be accomplished using known coupling techniques as described above. Ligation of an open circle probe hybridized to a tether loop is analogous to the ligation operation of LM-RCA. In this case, the target sequence is part of an oligonucleotide with both ends coupled to a specific binding molecule. This same ligation technique can be used to circularize open circle probes on target sequences that are part of reporter binding agents. This topologically locks the open circle probe to the reporter binding agent (and thus, to the target molecule to which the reporter binding agent binds).

[0202] The ends of tether loops can be coupled to any specific binding molecule with functional groups that can be derivatized with suitable activating groups. When the specific binding molecule is a protein, or a molecule with similar functional groups, coupling of tether ends can be accomplished using known methods of protein attachment. Many such methods are described in Protein immobilization: fundamentals and applications Richard F. Taylor, ed. (M. Dekker, New York, 1991).

[0203] Antibodies useful as the affinity portion of reporter binding agents, can be obtained commercially or produced using well established methods. For example, Johnstone and Thorpe, on pages 30-85, describe general methods useful for producing both polyclonal and monoclonal antibodies. The entire book describes many general techniques and principles for the use of antibodies in assay systems.

[0204] H. Detection Labels

[0205] To aid in detection and quantitation of nucleic acids amplified using the disclosed method, detection labels can be directly incorporated into amplified nucleic acids or can be coupled to detection molecules. As used herein, a detection label is any molecule that can be associated with amplified nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for incorporation into nucleic acids or coupling to nucleic acid or antibody probes are known to those of skill in the art. Examples of detection labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands. A preferred use of detection labels in the disclosed method is as a label in fluorescent change probes and primers.

[0206] Examples of suitable fluorescent labels include fluorescein (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4′-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine (5,6-tetramethyl rhodamine). Preferred fluorescent labels for combinatorial multicolor coding are FITC and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. The fluorescent labels can be obtained from a variety of commercial sources, including Molecular Probes, Eugene, Oreg. and Research Organics, Cleveland, Ohio.

[0207] Labeled nucleotides are preferred form of detection label since they can be directly incorporated into the products of RCA and RCT during synthesis. Examples of detection labels that can be incorporated into amplified DNA or RNA include nucleotide analogs such as BrdUrd (Hoy and Schimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364 (1992)). Suitable fluorescence-labeled nucleotides are Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotide analog detection label for DNA is BrdUrd (BUDR triphosphate, Sigma), and a preferred nucleotide analog detection label for RNA is Biotin-16-uridine-5′-triphosphate (Biotin-16-dUTP, Boehringher Mannheim). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling. Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes.

[0208] Detection labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[1,2,-dioxetane-3-2′-(5′-chloro)tricyclo [3.3.1.13,7]decane]-4-yl) phenyl phosphate; Tropix, Inc.).

[0209] A preferred detection label for use in detection of amplified RNA is acridinium-ester-labeled DNA probe (GenProbe, Inc., as described by Arnold et al., Clinical Chemistry 35:1588-1594 (1989)). An acridinium-ester-labeled detection probe permits the detection of amplified RNA without washing because unhybridized probe can be destroyed with alkali (Arnold et al. (1989)).

[0210] Molecules that combine two or more of these detection labels are also considered detection labels. Any of the known detection labels can be used with the disclosed probes, tags, and method to label and detect nucleic acid amplified using the disclosed method. Methods for detecting and measuring signals generated by detection labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary detection label coupled to the antibody. Such methods can be used directly in the disclosed method of amplification and detection. As used herein, detection molecules are molecules that interact with amplified nucleic acid and to which one or more detection labels are coupled. Fluorescent labels, especially in the context of fluorescent change probes and primers are useful for real-time detection of amplification.

[0211] I. Detection Probes

[0212] Detection probes are labeled oligonucleotides having sequence complementary to detection tags on TS-DNA or transcripts of TS-DNA. The complementary portion of a detection probe can be any length that supports specific and stable hybridization between the detection probe and the detection tag. For this purpose, a length of 10 to 35 nucleotides is preferred, with a complementary portion of a detection probe 16 to 20 nucleotides long being most preferred. Detection probes can contain any of the detection labels described above. Preferred labels are biotin and fluorescent molecules. Useful detection probes are fluorescent change probes. A particularly preferred detection probe is a molecular beacon (which is a form of fluorescent change probe). Molecular beacons are detection probes labeled with fluorescent moieties where the fluorescent moieties fluoresce only when the detection probe is hybridized (Tyagi and Kramer, Nature Biotechnology 14:303-308 (1996)). The use of such probes eliminates the need for removal of unhybridized probes prior to label detection because the unhybridized detection probes will not produce a signal. This is especially useful in multiplex assays.

[0213] One form of detection probe, referred to herein as a collapsing detection probe, contains two separate complementary portions. This allows each detection probe to hybridize to two detection tags in TS-DNA. In this way, the detection probe forms a bridge between different parts of the TS-DNA. The combined action of numerous collapsing detection probes hybridizing to TS-DNA will be to form a collapsed network of cross-linked TS-DNA. Collapsed TS-DNA occupies a much smaller volume than free, extended TS-DNA, and includes whatever detection label present on the detection probe. This result is a compact and discrete detectable signal for each TS-DNA. Collapsing TS-DNA is useful both for in situ hybridization applications and for multiplex detection because it allows detectable signals to be spatially separate even when closely packed. Collapsing TS-DNA is especially preferred for use with combinatorial multicolor coding.

[0214] TS-DNA collapse can also be accomplished through the use of ligand/ligand binding pairs (such as biotin and avidin) or hapten/antibody pairs. As described in U.S. Pat. No. 6,143,495 (Example 6), a nucleotide analog, BUDR, can be incorporated into TS-DNA during rolling circle replication. When biotinylated antibodies specific for BUDR and avidin are added, a cross-linked network of TS-DNA forms, bridged by avidin-biotin-antibody conjugates, and the TS-DNA collapses into a compact structure. Collapsing detection probes and biotin-mediated collapse can also be used together to collapse TS-DNA.

[0215] J. Address Probes

[0216] An address probe is an oligonucleotide having a sequence complementary to address tags on TS-DNA or transcripts of TS-DNA. The complementary portion of an address probe can be any length that supports specific and stable hybridization between the address probe and the address tag. For this purpose, a length of 10 to 35 nucleotides is preferred, with a complementary portion of an address probe 12 to 18 nucleotides long being most preferred. Preferably, the complementary portion of an address probe is complementary to all or a portion of the target probe portions of an OCP. Most preferably, the complementary portion of an address probe is complementary to a portion of either or both of the left and right target probe portions of an OCP and all or a part of any gap oligonucleotides or gap sequence created in a gap-filling operation (see FIG. 6 of U.S. Pat. No. 6,143,495). Address probe can contain a single complementary portion or multiple complementary portions. Preferably, address probes are coupled, either directly or via a spacer molecule, to a solid-state support. Such a combination of address probe and solid-state support are a preferred form of solid-state detector. Address probes can be fluorescent change probes although this is not preferred.

[0217] K. Oligonucleotide Synthesis

[0218] Methods to produce or synthesize oligonucleotides are well known in the art. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method. Solid phase chemical synthesis of DNA fragments is routinely performed using protected nucleoside cyanoethyl phosphoramidites (S. L. Beaucage et al. (1981) Tetrahedron Lett. 22:1859). In this approach, the 3′-hydroxyl group of an initial 5′-protected nucleoside is first covalently attached to the polymer support (R. C. Pless et al. (1975) Nucleic Acids Res. 2:773 (1975)). Synthesis of the oligonucleotide then proceeds by deprotection of the 5′-hydroxyl group of the attached nucleoside, followed by coupling of an incoming nucleoside-3′-phosphoramidite to the deprotected hydroxyl group (M. D. Matteucci et a. (1981) J. Am. Chem. Soc. 103:3185). The resulting phosphite triester is finally oxidized to a phosphorotriester to complete the internucleotide bond (R. L. Letsinger et al. (1976) J. Am. Chem. Soc. 9:3655). Alternatively, the synthesis of phosphorothioate linkages can be carried out by sulfurization of the phosphite triester. Several chemicals can be used to perform this reaction, among them 3H-1,2-benzodithiole-3-one, 1,1-dioxide (R. P. Iyer, W. Egan, J. B. Regan, and S. L. Beaucage, J. Am. Chem. Soc., 1990, 112, 1253-1254). The steps of deprotection, coupling and oxidation are repeated until an oligonucleotide of the desired length and sequence is obtained. Other methods exist to generate oligonucleotides such as the H-phosphonate method (Hall et al, (1957) J. Chem. Soc., 3291-3296) or the phosphotriester method as described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994). Other forms of oligonucleotide synthesis are described in U.S. Pat. Nos. 6,294,664 and 6,291,669.

[0219] The nucleotide sequence of an oligonucleotide is generally determined by the sequential order in which subunits of subunit blocks are added to the oligonucleotide chain during synthesis. Each round of addition can involve a different, specific nucleotide precursor, or a mixture of one or more different nucleotide precursors. In general, degenerate or random positions in an oligonucleotide can be produced by using a mixture of nucleotide precursors representing the range of nucleotides that can be present at that position. Thus, precursors for A and T can be included in the reaction for a particular position in an oligonucleotide if that position is to be degenerate for A and T. Precursors for all four nucleotides can be included for a fully degenerate or random position. Completely random oligonucleotides can be made by including all four nucleotide precursors in every round of synthesis. Degenerate oligonucleotides can also be made having different proportions of different nucleotides. Such oligonucleotides can be made, for example, by using different nucleotide precursors, in the desired proportions, in the reaction.

[0220] Many of the oligonucleotides described herein are designed to be complementary to certain portions of other oligonucleotides or nucleic acids such that stable hybrids can be formed between them. The stability of these hybrids can be calculated using known methods such as those described in Lesnick and Freier, Biochemistry 34:10807-10815 (1995), McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al., Nucleic Acids Res. 18:6409-6412 (1990).

[0221] As an example, random oligonucleotides can be synthesized on a Perseptive Biosystems 8909 Expedite Nucleic Acid Synthesis system using standard &bgr;-cyanoethyl phosphoramidite coupling chemistry on mixed dA+dC+dG+dT synthesis columns (Glen Research, Sterling, Va.). The four phosphoramidites can be mixed in equal proportions to randomize the bases at each position in the oligonucleotide. Oxidation of the newly formed phosphites can be carried out using the sulfurizing reagent 3H-1,2-benzothiole-3-one-1,1-idoxide (Glen Research) instead of the standard oxidizing reagent after the first and second phosphoramidite addition steps. The thio-phosphitylated oligonucleotides can be deprotected using 30% ammonium hydroxide (3.0 ml) in water at 55° C. for 16 hours, concentrated in an OP 120 Savant Oligo Prep deprotection unit for 2 hours, and desalted with PD10 Sephadex columns using the protocol provided by the manufacturer.

[0222] Open circle probes, fluorescent change probe and primers, amplification target circles, rolling circle replication primers, detection probes, address probes, DNA strand displacement primers, and any other oligonucleotides can be synthesized using established oligonucleotide synthesis methods. Methods to produce or synthesize oligonucleotides are well known in the art. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method. Solid phase chemical synthesis of DNA fragments is routinely performed using protected nucleoside cyanoethyl phosphoramidites (S. L. Beaucage et al. (1981) Tetrahedron Lett. 22:1859). In this approach, the 3′-hydroxyl group of an initial 5′-protected nucleoside is first covalently attached to the polymer support (R. C. Pless et al. (1975) Nucleic Acids Res. 2:773 (1975)). Synthesis of the oligonucleotide then proceeds by deprotection of the 5′-hydroxyl group of the attached nucleoside, followed by coupling of an incoming nucleoside-3′-phosphoramidite to the deprotected hydroxyl group (M. D. Matteucci et a. (1981) J. Am. Chem. Soc. 103:3185). The resulting phosphite triester is finally oxidized to a phosphorotriester to complete the internucleotide bond (R. L. Letsinger et al. (1976) J. Am. Chem. Soc. 9:3655). Alternatively, the synthesis of phosphorothioate linkages can be carried out by sulfurization of the phosphite triester. Several chemicals can be used to perform this reaction, among them 3H-1,2-benzodithiole-3-one, 1,1-dioxide (R. P. Iyer, W. Egan, J. B. Regan, and S. L. Beaucage, J. Am. Chem. Soc., 1990, 112, 1253-1254). The steps of deprotection, coupling and oxidation are repeated until an oligonucleotide of the desired length and sequence is obtained. Other methods exist to generate oligonucleotides such as the H-phosphonate method (Hall et al, (1957) J. Chem. Soc., 3291-3296) or the phosphotriester method as described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994). Other forms of oligonucleotide synthesis are described in U.S. Pat. Nos. 6,294,664 and 6,291,669.

[0223] Many of the oligonucleotides described herein are designed to be complementary to certain portions of other oligonucleotides or nucleic acids such that stable hybrids can be formed between them via base pairing. The stability of these hybrids can be calculated using known methods such as those described in Lesnick and Freier, Biochemistry 34:10807-10815 (1995), McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al., Nucleic Acids Res. 18:6409-6412 (1990).

[0224] Oligonucleotides can be synthesized, for example, on a Perseptive Biosystems 8909 Expedite Nucleic Acid Synthesis system using standard &bgr;-cyanoethyl phosphoramidite coupling chemistry on synthesis columns (Glen Research, Sterling, Va.). Oxidation of the newly formed phosphites can be carried out using, for example, the sulfurizing reagent 3H-1,2-benzothiole-3-one-1,1-idoxide (Glen Research) or the standard oxidizing reagent after the first and second phosphoramidite addition steps. The thio-phosphitylated oligonucleotides can be deprotected, for example, using 30% ammonium hydroxide (3.0 ml) in water at 55° C. for 16 hours, concentrated in an OP 120 Savant Oligo Prep deprotection unit for 2 hours, and desalted with PD 10 Sephadex columns using the protocol provided by the manufacturer.

[0225] So long as their relevant function is maintained, open circle probes, fluorescent change probe and primers, amplification target circles, rolling circle replication primers, detection probes, address probes, DNA strand displacement primers, and any other oligonucleotides can be made up of or include modified nucleotides (nucleotide analogs). Many modified nucleotides are known and can be used in oligonucleotides. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includes but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Other modified bases are those that function as universal bases. Universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases substitute for the normal bases but have no bias in base pairing. That is, universal bases can base pair with any other base. Base modifications often can be combined with for example a sugar modification, such as 2′-O-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference in its entirety, and specifically for their description of base modifications, their synthesis, their use, and their incorporation into oligonucleotides and nucleic acids.

[0226] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxyribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl and alkynyl. 2′ sugar modifications also include but are not limited to —O[(CH2)n O]m CH3, —O(CH2)n OCH3, —O(CH2)n NH2, —O(CH2)n CH3, —O(CH2)n —ONH2, and —O(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10.

[0227] Other modifications at the 2′ position include but are not limited to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety, and specifically for their description of modified sugar structures, their synthesis, their use, and their incorporation into nucleotides, oligonucleotides and nucleic acids.

[0228] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3′-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkages between two nucleotides can be through a 3′-5′ linkage or a 2′-5′ linkage, and the linkage can contain inverted polarity such as 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference its entirety, and specifically for their description of modified phosphates, their synthesis, their use, and their incorporation into nucleotides, oligonucleotides and nucleic acids.

[0229] It is understood that nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.

[0230] Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize and hybridize to (base pair to) complementary nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

[0231] Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference its entirety, and specifically for their description of phosphate replacements, their synthesis, their use, and their incorporation into nucleotides, oligonucleotides and nucleic acids.

[0232] It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al., Science 254:1497-1500 (1991)).

[0233] Oligonucleotides can be comprised of nucleotides and can be made up of different types of nucleotides or the same type of nucleotides. For example, one or more of the nucleotides in an oligonucleotide can be ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2′-O-methyl ribonucleotides; about 10% to about 50% of the nucleotides can be ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2′-O-methyl ribonucleotides; about 50% or more of the nucleotides can be ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2′-O-methyl ribonucleotides; or all of the nucleotides are ribonucleotides, 2′-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2′-O-methyl ribonucleotides. Such oligonucleotides can be referred to as chimeric oligonucleotides.

[0234] L. Solid-State Detectors

[0235] Solid-state detectors are solid-state substrates or supports to which address probes or detection molecules have been coupled. A preferred form of solid-state detector is an array detector. An array detector is a solid-state detector to which multiple different address probes or detection molecules have been coupled in an array, grid, or other organized pattern.

[0236] Solid-state substrates for use in solid-state detectors can include any solid material to which oligonucleotides can be coupled. This includes materials such as acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, particles and microparticles. A chip is a rectangular or square small piece of material. Preferred forms for solid-state substrates are thin films, beads, or chips.

[0237] Address probes immobilized on a solid-state substrate allow capture of the products of the disclosed amplification method on a solid-state detector. Such capture provides a convenient means of washing away reaction components that might interfere with subsequent detection steps. By attaching different address probes to different regions of a solid-state detector, different amplification products can be captured at different, and therefore diagnostic, locations on the solid-state detector. For example, in a multiplex assay, address probes specific for numerous different amplified nucleic acids (each representing a different target sequence amplified via a different set of primers) can be immobilized in an array, each in a different location. Capture and detection will occur only at those array locations corresponding to amplified nucleic acids for which the corresponding target sequences were present in a sample.

[0238] Methods for immobilization of oligonucleotides to solid-state substrates are well established. Oligonucleotides, including address probes and detection probes, can be coupled to substrates using established coupling methods. For example, suitable attachment methods are described by Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and Khrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method for immobilization of 3′-amine oligonucleotides on casein-coated slides is described by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383 (1995). A preferred method of attaching oligonucleotides to solid-state substrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465 (1994). Examples of nucleic acid chips and arrays, including methods of making and using such chips and arrays, are described in U.S. Pat. Nos. 6,287,768, 6,288,220, 6,287,776, 6,297,006, and 6,291,193.

[0239] Some solid-state detectors useful in the disclosed method have detection antibodies attached to a solid-state substrate. Such antibodies can be specific for a molecule of interest. Captured molecules of interest can then be detected by binding of a second, reporter antibody, followed by amplification. Such a use of antibodies in a solid-state detector allows amplification assays to be developed for the detection of any molecule for which antibodies can be generated. Methods for immobilizing antibodies to solid-state substrates are well established. Immobilization can be accomplished by attachment, for example, to aminated surfaces, carboxylated surfaces or hydroxylated surfaces using standard immobilization chemistries. Examples of attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents, epoxides and maleimides. A preferred attachment agent is glutaraldehyde. These and other attachment agents, as well as methods for their use in attachment, are described in Protein immobilization: fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, New York, 1991), Johnstone and Thorpe, Immunochemistry In Practice (Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216 and 241-242, and Immobilized Affinity Ligands, Craig T. Hermanson et al., eds. (Academic Press, New York, 1992). Antibodies can be attached to a substrate by chemically cross-linking a free amino group on the antibody to reactive side groups present within the solid-state substrate. For example, antibodies may be chemically cross-linked to a substrate that contains free amino or carboxyl groups using glutaraldehyde or carbodiimides as cross-linker agents. In this method, aqueous solutions containing free antibodies are incubated with the solid-state substrate in the presence of glutaraldehyde or carbodiimide. For crosslinking with glutaraldehyde the reactants can be incubated with 2% glutaraldehyde by volume in a buffered solution such as 0.1 M sodium cacodylate at pH 7.4. Other standard immobilization chemistries are known by those of skill in the art.

[0240] M. Solid-State Samples

[0241] Solid-state samples are solid-state substrates or supports to which target molecules or target sequences have been coupled or adhered. Target molecules or target sequences are preferably delivered in a target sample or assay sample. A preferred form of solid-state sample is an array sample. An array sample is a solid-state sample to which multiple different target samples or assay samples have been coupled or adhered in an array, grid, or other organized pattern.

[0242] Solid-state substrates for use in solid-state samples can include any solid material to which target molecules or target sequences can be coupled or adhered. This includes materials such as acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, particles and microparticles. A chip is a rectangular or square small piece of material. Preferred forms for solid-state substrates are thin films, beads, or chips.

[0243] Target molecules and target sequences immobilized on a solid-state substrate allow formation of target-specific TS-DNA localized on the solid-state substrate. Such localization provides a convenient means of washing away reaction components that might interfere with subsequent detection steps, and a convenient way of assaying multiple different samples simultaneously. Diagnostic TS-DNA can be independently formed at each site where a different sample is adhered. For immobilization of target sequences or other oligonucleotide molecules to form a solid-state sample, the methods described above for can be used. Nucleic acids produced in the disclosed method can be coupled or adhered to a solid-state substrate in any suitable way. For example, nucleic acids generated by multiple strand displacement can be attached by adding modified nucleotides to the 3′ ends of nucleic acids produced by strand displacement replication using terminal deoxynucleotidyl transferase, and reacting the modified nucleotides with a solid-state substrate or support thereby attaching the nucleic acids to the solid-state substrate or support.

[0244] A preferred form of solid-state substrate is a glass slide to which up to 256 separate target samples have been adhered as an array of small dots. Each dot is preferably from 0.1 to 2.5 mm in diameter, and most preferably around 2.5 mm in diameter. Such microarrays can be fabricated, for example, using the method described by Schena et al., Science 270:487-470 (1995). Briefly, microarrays can be fabricated on poly-L-lysine-coated microscope slides (Sigma) with an arraying machine fitted with one printing tip. The tip is loaded with 1 &mgr;l of a DNA sample (0.5 mg/ml) from, for example, 96-well microtiter plates and deposited ˜0.005 &mgr;l per slide on multiple slides at the desired spacing. The printed slides can then be rehydrated for 2 hours in a humid chamber, snap-dried at 100° C. for 1 minute, rinsed in 0.1% SDS, and treated with 0.05% succinic anhydride prepared in buffer consisting of 50% 1-methyl-2-pyrrolidinone and 50% boric acid. The DNA on the slides can then be denatured in, for example, distilled water for 2 minutes at 90° C. immediately before use. Microarray solid-state samples can scanned with, for example, a laser fluorescent scanner with a computer-controlled XY stage and a microscope objective. A mixed gas, multiline laser allows sequential excitation of multiple fluorophores.

[0245] N. DNA ligases

[0246] Any DNA ligase is suitable for use in the disclosed amplification method. Preferred ligases are those that preferentially form phosphodiester bonds at nicks in double-stranded DNA. That is, ligases that fail to ligate the free ends of single-stranded DNA at a significant rate are preferred. Thermostable ligases are especially preferred. Many suitable ligases are known, such as T4 DNA ligase (Davis et al., Advanced Bacterial Genetics—A Manual for Genetic Engineering (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1980)), E. coli DNA ligase (Panasnko et al., J. Biol. Chem. 253:4590-4592 (1978)), AMPLIGASE® (Kalin et al., Mutat. Res., 283(2):119-123 (1992); Winn-Deen et al., Mol Cell Probes (England) 7(3):179-186 (1993)), Taq DNA ligase (Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991), Thermus thermophilus DNA ligase (Abbott Laboratories), Thermus scotoductus DNA ligase and Rhodothermus marinus DNA ligase (Thorbjarnardottir et al., Gene 151:177-180 (1995)). T4 DNA ligase is preferred for ligations involving RNA target sequences due to its ability to ligate DNA ends involved in DNA:RNA hybrids (Hsuih et al., Quantitative detection of HCV RNA using novel ligation-dependent polymerase chain reaction, American Association for the Study of Liver Diseases (Chicago, Ill., Nov. 3-7, 1995)).

[0247] The frequency of non-target-directed ligation catalyzed by a ligase can be determined as follows. LM-RCA is performed with an open circle probe and a gap oligonucleotide in the presence of a target sequence. Non-targeted-directed ligation products can then be detected by using an address probe specific for the open circle probe ligated without the gap oligonucleotide to capture TS-DNA from such ligated probes. Target directed ligation products can be detected by using an address probe specific for the open circle probe ligated with the gap oligonucleotide. By using a solid-state detector with regions containing each of these address probes, both target directed and non-target-directed ligation products can be detected and quantitated. The ratio of target-directed and non-target-directed TS-DNA produced provides a measure of the specificity of the ligation operation. Target-directed ligation can also be assessed as discussed in Barany (1991).

[0248] O. DNA Polymerases

[0249] DNA polymerases useful in the rolling circle replication step of the disclosed method must perform rolling circle replication of primed single-stranded circles. Such polymerases are referred to herein as rolling circle DNA polymerases. For rolling circle replication, it is preferred that a DNA polymerase be capable of displacing the strand complementary to the template strand, termed strand displacement, and lack a 5′ to 3′ exonuclease activity. Strand displacement is necessary to result in synthesis of multiple tandem copies of the ligated OCP. A 5′ to 3′ exonuclease activity, if present, might result in the destruction of the synthesized strand. DNA polymerases for use in the disclosed method can also be highly processive, if desired. The suitability of a DNA polymerase for use in the disclosed method can be readily determined by assessing its ability to carry out rolling circle replication. Preferred rolling circle DNA polymerases are Bst DNA polymerase, VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)), ThermoSequenase™, delta Tts DNA polymerase, Bea DNA polymerase (Journal of Biochemistry 113(3):401-10, 1993 Mar.), bacteriophage &phgr;29 DNA polymerase (U.S. Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNA polymerase (Matsumoto et al., Gene 84:247 (1989)), phage &phgr;PRD1 DNA polymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19 (1991)), PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), modified T7 DNA polymerase (Tabor and Richardson, J. Biol. Chem. 262:15330-15333 (1987); Tabor and Richardson, J. Biol. Chem. 264:6447-6458 (1989); Sequenase™ (U.S. Biochemicals)), and T4 DNA polymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157 (1995)). More preferred are Bst DNA polymerase, VENT® DNA polymerase, ThermoSequenase™, and delta Tts DNA polymerase. Bst DNA polymerase is most preferred.

[0250] Strand displacement can be facilitated through the use of a strand displacement factor, such as helicase. It is considered that any DNA polymerase that can perform rolling circle replication in the presence of a strand displacement factor is suitable for use in the disclosed method, even if the DNA polymerase does not perform rolling circle replication in the absence of such a factor. Strand displacement factors useful in the disclosed method include BMRFI polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA binding-proteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)).

[0251] The ability of a polymerase to carry out rolling circle replication can be determined by using the polymerase in a rolling circle replication assay such as those described in Fire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995) and in U.S. Pat. No. 6,143,495 (Example 1).

[0252] Another type of DNA polymerase can be used if a gap-filling synthesis step is used, such as in gap-filling LM-RCA (see U.S. Pat. No. 6,143,495, Example 3). When using a DNA polymerase to fill gaps, strand displacement by the DNA polymerase is undesirable. Such DNA polymerases are referred to herein as gap-filling DNA polymerases. Unless otherwise indicated, a DNA polymerase referred to herein without specifying it as a rolling circle DNA polymerase or a gap-filling DNA polymerase, is understood to be a rolling circle DNA polymerase and not a gap-filling DNA polymerase. Preferred gap-filling DNA polymerases are T7 DNA polymerase (Studier et al., Methods Enzymol. 185:60-89 (1990)), DEEP VENTS DNA polymerase (New England Biolabs, Beverly, Mass.), modified T7 DNA polymerase (Tabor and Richardson, J. Biol. Chem. 262:15330-15333 (1987); Tabor and Richardson, J. Biol. Chem. 264:6447-6458 (1989); Sequenase™ (U.S. Biochemicals)), and T4 DNA polymerase (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). An especially preferred type of gap-filling DNA polymerase is the Thermus flavus DNA polymerase (MBR, Milwaukee, Wis.). The most preferred gap-filling DNA polymerase is the Stoffel fragment of Taq DNA polymerase (Lawyer et al., PCR Methods Appl. 2(4):275-287 (1993), King et al., J. Biol. Chem. 269(18):13061-13064 (1994)).

[0253] The ability of a polymerase to fill gaps can be determined by performing gap-filling LM-RCA. Gap-filling LM-RCA is performed with an open circle probe that forms a gap space when hybridized to the target sequence. Ligation can only occur when the gap space is filled by the DNA polymerase. If gap-filling occurs, TS-DNA can be detected, otherwise it can be concluded that the DNA polymerase, or the reaction conditions, is not useful as a gap-filling DNA polymerase.

[0254] P. RNA polymerases

[0255] Any RNA polymerase which can carry out transcription in vitro and for which promoter sequences have been identified can be used in the disclosed rolling circle transcription method. Stable RNA polymerases without complex requirements are preferred. Most preferred are T7 RNA polymerase (Davanloo et al., Proc. Natl. Acad. Sci. USA 81:2035-2039 (1984)) and SP6 RNA polymerase (Butler and Chamberlin, J. Biol. Chem. 257:5772-5778 (1982)) which are highly specific for particular promoter sequences (Schenbom and Meirendorf, Nucleic Acids Research 13:6223-6236 (1985)). Other RNA polymerases with this characteristic are also preferred. Because promoter sequences are generally recognized by specific RNA polymerases, the OCP or ATC should contain a promoter sequence recognized by the RNA polymerase that is used. Numerous promoter sequences are known and any suitable RNA polymerase having an identified promoter sequence can be used. Promoter sequences for RNA polymerases can be identified using established techniques.

[0256] Q. Kits

[0257] The materials described above can be packaged together in any suitable combination as a kit useful for performing the disclosed method. All such possible combinations are specifically contemplated. It is preferred that the kit components in a given kit be designed and adapted for use together in the disclosed method. A kit can include, for example, one or more open circle probes and one or more detection rolling circle replication primers. A kit can also include a secondary DNA strand displacement primer, a common rolling circle replication primer, or both. The open circle probes, detection rolling circle replication primers, common rolling circle replication primers and secondary DNA strand displacement primers.

[0258] A kit can include, for example, a set of open circle probes each comprising two ends, where at least one of the ends of one of the open circle probe can form an intramolecular stem structure, where portions of each open circle probe are complementary to the one or more target sequences, and a plurality of detection rolling circle replication primers, where all or a portion of each detection rolling circle replication primer is complementary to a portion of one or more of the open circle probes. Such kits can also include, for example, one or more secondary DNA strand displacement primers, where all or a portion of each secondary DNA strand displacement primer matches a portion of one or more of the open circle probes, and one or more common rolling circle replication primers, where all or a portion of each common rolling circle replication primer is complementary to a portion of one or more of the open circle probes.

[0259] A kit can also include one or more gap oligonucleotides. The target probe portions of the open circle probes in a kit preferably are each complementary to a different target sequence or to different forms of the same target sequence. A kit can also include one or more detection probes. Preferably, a portion of each of the detection probes in a kit has sequence matching or complementary to a portion of a different one of the open circle probes in that kit.

[0260] A kit can also include one or more reporter binding agents where the oligonucleotide portion of the reporter binding agents include one of the target-sequences. The specific binding molecules of the reporter binding agents in a kit each can be specific for an analyte, preferably specific for a protein or peptide.

Method

[0261] The disclosed method involves an amplification operations and, optionally, a ligation operation. The method provides nucleic acid amplification reactions that reduce, prevent, or eliminate artifacts; increase efficiency; increase specificity; and/or increase consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers.

[0262] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation. The DNA ligation operation can comprise circularization of one or more open circle probes and can be carried out in the presence of a set of open circle probes. The set of open circle probes can comprise a plurality of different open circle probes. Each open circle probe can comprise two ends, where at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure can be dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set.

[0263] The amplification operation can comprise rolling circle replication of the circularized open circle probes. The amplification operation can be carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in the set of open circle probes.

[0264] Some forms of the disclosed method can comprise an amplification operation. The amplification operation can be carried out in the presence of a set of amplification target circles. The set of amplification target circles can comprise a plurality of different amplification target circles. The amplification operation can comprise rolling circle replication of the amplification target circles. The amplification operation can be carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer can correspond to a different amplification target circle in the set of amplification target circles, the secondary DNA strand displacement primer can correspond to all of the amplification target circles in the set of amplification target circles, and the common rolling circle replication primer can correspond to all of the amplification target circles in the set of amplification target circles.

[0265] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes, and where two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0266] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes. The amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, where each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

[0267] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and where the amplification operation comprises rolling circle replication of the circularized open circle probes. The amplification operation is carried out in the presence of one or more rolling circle replication primers, where at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher.

[0268] Some forms of the disclosed method can comprise an amplification operation, where the amplification operation comprises rolling circle replication of the amplification target circles. The amplification operation is carried out in the presence of one or more rolling circle replication primers, where at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher.

[0269] Some forms of the disclosed method can comprise a DNA ligation operation and an amplification operation, where the DNA ligation operation comprises circularization of one or more open circle probes, and wherein the amplification operation comprises rolling circle replication of the circularized open circle probes. The ligation operation is carried out in the presence of a set of open circle probes, where the set of open circle probes comprises a plurality of different open circle probes. Each open circle probe comprises two ends, where at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0270] The amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer is associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

[0271] Some forms of the disclosed method can comprise

[0272] (a) mixing a set of open circle probes with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture. The set of open circle probes comprises a plurality of different open circle probes. Each open circle probe can comprise two ends. At least one of the ends of at least one of the open circle probes can form an intramolecular stem structure. Circularization of the open circle probes that can form an intramolecular stem structure can be dependent on hybridization of the open circle probe to a target sequence. Two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set.

[0273] (b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles. The amplification target circles formed from the open circle probes in the set of open circle probes can comprise a set of amplification target circles.

[0274] (c) mixing a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher. Each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, the secondary DNA strand displacement primer can correspond to all of the open circle probes in the set of open circle probes, and the common rolling circle replication primer can correspond to all of the open circle probes in the set of open circle probes.

[0275] (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles. Replication of the amplification target circles results in the formation of tandem sequence DNA.

[0276] Some forms of the disclosed compositions and method can increase the efficiency of nucleic acid amplification reactions. Increased efficiency can include, for example, increased amplification and/or signal generation in less time, from less starting material, and/or from less reagents; and/or signal detection during the amplification reaction. One form of method for increasing efficiency is the use of a detection primer, such as a detection rolling circle replication primer. The detection primer produces a signal during amplification as a quenching moiety in or on the primer becomes separated from a fluorescent label on the primer. A useful form of detection primer is a detection rolling circle primer associated with a peptide nucleic acid quencher. The peptide nucleic acid quencher is displaced from the detection primer as amplification proceeds (via, for example, replication of a nucleic acid strand complementary to the nucleic acid strand that incorporates the primer).

[0277] The progress of rolling circle amplification reactions can be monitored in real-time (that is, during the reaction) by using detection primers in the amplification. The detection primer produces a signal during amplification as a quenching moiety in or on the primer becomes separated from a fluorescent label on the primer. When a quenching moiety is in proximity to a fluorescent molecule or label, fluorescence is quenched by transfer of energy to the quenching moiety. Fluorescence is detectable once the quenching moiety is no longer in proximity to the fluorescent label. Detection primers are incorporated into amplification products as they prime replication. In the disclosed amplification reactions, the incorporated primer goes on to serve as a template sequence when the nucleic acid strand in which it is incorporated is replicated. A quenching moiety can be placed in proximity to a fluorescent label on the primer, for example, via hybridization of a nucleic acid sequence to which the quenching moiety is attached to sequence of the primer adjacent to the fluorescent label. When the incorporated primer is replicated, the hybrid is disrupted and the quencher moiety is separated from the fluorescent label, which can then produce a fluorescent signal. Thus, as the amplification reaction proceeds, more and more incorporated detection primers are replicated, producing an ever-increasing fluorescent signal that can be monitored as the reaction proceeds.

[0278] Another form of method for increasing efficiency is the use of combinations of primers having different relationships to open circle probes used in the method. For example, the use of two or more rolling circle replication primers and one or more secondary DNA strand displacement primers, with each primer specific for a different sequence or region of the open circle probes, can increase the efficiency of amplification by producing multiple simultaneous initiations of replication and multiple simultaneous generations of amplification product simultaneously. For example, each of two or more different rolling circle replication primers can simultaneously prime replication from different sequences in a given circularized open circle probe or amplification target circle. This multiplies the yield of amplification.

[0279] Rolling circle amplification involves rolling circle replication of a circular template, such as a circularized open circle probe or an amplification target circle. Rolling circle replication can be mediated by a primer, referred to as a rolling circle replication primer, that hybridizes anywhere on the circular temple. Multiple strands can be produced simultaneously by using two or more rolling circle replication primers that hybridize to different sequences (that is, at different locations) in the circular template. Thus, the disclosed method can be performed using of two or more rolling circle replication primers targeted to different sequences in the circular templates. Particularly useful are the use of detection rolling circle replication primers and common rolling circle replication primers in amplification reactions where both a detection rolling circle replication primer and a common rolling circle replication primer correspond to each open circle probe or amplification target circle.

[0280] Use of both rolling circle replication primers (which prime replication of circularized open circle probes and amplification target circles) and secondary DNA strand displacement primers (which prime replication of the product of replication of circularized open circle probes and amplification target circles) allows multiple generations of amplification product to be generated simultaneously. This multiplies the yield of amplification.

[0281] Rolling circle replication of a circular template produces long strands of DNA containing tandem repeats of sequence complementary to the sequence of the circular template. These strands are referred to as tandem sequence DNA. The speed and yield of rolling circle amplification reactions can be greatly increased by replicating the tandem sequence DNA during rolling circle replication. This can be accomplished by using one or more primers complementary to sequence in the tandem sequence DNA. Such primers, referred to as secondary DNA strand displacement primers, have sequence matching sequence in an open circle probe or amplification target circle (and thus are complementary to the tandem sequence DNA). Replication of the tandem sequence DNA produces more nucleic acid, referred to as secondary tandem sequence DNA, and provides a template for further replication by the rolling circle replication primers (which are complementary to sequences in the secondary tandem sequence DNA). These, and subsequent replication products are similarly replicated producing an overall cascade of replication, referred to as exponential rolling circle amplification, that produces a huge amplification in a short time.

[0282] Some forms of the disclosed compositions and method can increase the specificity of nucleic acid amplification reactions. Increased specificity can include, for example, more amplification of amplification targets, or more amplification based on specific targets, relative to non-target amplification and/or more accurate assessment of false positive and false negative amplification. One form of method for increasing specificity is the use of matched open circle probe sets. Matched open circle probes are open circle probes that are targeted to different forms of the same target sequence. For example, a target sequence in a gene of interest may occur in two or more forms (for example, a “wild type” or “normal” form and a “mutant” form; or, more generally, polymorphic forms); single nucleotide polymorphisms are an example of such different forms of target sequences. By targeting two or more (up to, for example, most or all) of the different forms of a target sequence that may be present, the amplification reaction will include a positive control. That is, for example, the open circle probe targeted to the normal form of the target sequence will produce a signal even if the mutant form of the target sequence is not present in the reaction or the open circle probe targeted to the mutant form of the target sequence will produce a signal even if the normal form of the target sequence is not present in the reaction.

[0283] Ligation-mediated rolling circle amplification should produce amplification products from a given open circle probe when the target sequence of that open circle probe is present and should not produce amplification products from that open circle probe when the target sequence of that open circle probe is not present. However, it is possible that the absence of the amplification products could be the result of a non-functional reaction rather than the absence of target sequence. Including open target circles specific for two or more possible forms of a target sequence increases the chance that the target for at least one of the open circle probes will be present. Resultant production of amplification products serves as a sort of positive control, indicating that the amplification reaction is functional. Further, if there is no target sequence present in the reaction (so that no open circle probe should be circularized and amplified), there is an increased tendency for the reaction to produce spurious or artifactual amplification products. This can be referred to as idle assay artifact production. By ensuring (or increasing the chances) that the target sequence for at least one open circle is present in the amplification reaction, the chance that idle assay artifacts will be produced.

[0284] Some forms of the disclosed compositions and method can increase the consistency of nucleic acid amplification reactions. Increased consistency can include, for example, levels of amplification products that more accurately reflect the relative amount of starting material, and/or less variation in the yield of amplification from different amplification targets. One form of method for increasing consistency involves the use of three primers having different relationships to open circle probes used in the method. The three primers are detection rolling circle replication primers, secondary DNA strand displacement primers, and common rolling circle replication primers. For example, for a given set of open circle probes or amplification target circles, detection rolling circle replication primers can each correspond to a different open circle probe or amplification target circle in the set while secondary DNA strand displacement primers and common rolling circle replication primers can correspond to all of the open circle probe or amplification target circles in the set. These relationships allow the overall amplification to be consistent among different open circle probes or amplification target circles in a set because the sequence of two of the primers used (and their complements on the circles) will be the same throughout the set (thus minimizing or eliminating the effect of sequence on primer efficiency). Differential detection is mediated by the circle-specific detection rolling circle replication primers.

[0285] Rolling circle amplification can be performed using multiple open circle probes or amplification target circles in the same reaction. Specificity of detection of rolling circle replication of different circularized open circle probes and amplification target circles can be accomplished in numerous ways. For real-time detection, it is useful to use a different detection rolling circle replication primer specific for each different open circle probe and amplification target circle. Because the different detection rolling circle replication primers may have different priming efficiencies (due to sequence differences, for example), it is useful to include one or more common rolling circle replication primers that are complementary to all of the open circle probes or amplification target circles in the reaction. This provides rolling circle replication unbiased by differing priming efficiencies.

[0286] Some forms of the disclosed compositions and method can reduce or eliminate generation of unwanted, undesirable, or non-specific amplification products in nucleic acid amplification reactions. One form of composition is an open circle probe that can form an intramolecular stem structure, such as a hairpin structure, at one or both ends. Open circle probes are useful in rolling circle amplification techniques. The stem structure allows the open circle probe to be circularized when hybridized to a legitimate target sequence but results in inactivation of uncircularized open circle probes. This inactivation, which preferably involves stabilization of the stem structure, extension of the end of the open circle probe, or both, reduces or eliminates the ability of the open circle probe to prime nucleic acid synthesis or to serve as a template for rolling circle amplification.

[0287] In ligation-mediated rolling circle amplification (LM-RCA), a linear DNA molecule, referred to as an open circle probe or padlock probe, hybridizes to a target sequence and is circularized. The circularized probe is then amplified via rolling circle replication of the circular probe. Uncircularized probe that remains in the reaction can hybridize to nucleic acid sequences in the reaction and cause amplification of undesirable, non-specific sequences. The disclosed compositions and method address this problem by reducing or eliminating the potential uncircularized open circle probes from priming nucleic acid synthesis.

[0288] The disclosed open circle probes can be inactivated in several ways. For example, where the 3′ end of an open circle probe is involved in an intramolecular stem structure, the 3′ end can be extended in a replication reaction using the open circle probe sequences as template (see FIG. 2B). The result is stabilization of the intramolecular stem structure and a change in the 3′ end sequence. Stabilization of the stem structure results in a reduction or elimination of the ability of the open circle probe to prime nucleic acid synthesis because the 3′ end is stably hybridized to sequences in the open circle probe under the conditions used for nucleic acid replication. Change in the sequence of the 3′ end can reduce of the ability of the open circle probe to prime nucleic acid synthesis because the changed 3′ sequences may not be as closely related to sequences involved in the amplification reaction or assay. Change in the sequence of the 3′ end can reduce of the ability of the open circle probe to serve as a template for rolling circle amplification. For example, even if the open circle probe with extended 3′ end were circularized, the rolling circle replication primer could be prevented from priming replication of such a circle if the primer complement sequence on the open circle probe were interrupted by the added sequences. This can be accomplished by, for example, designing the open circle probe to have the primer complement sequence include both 5′ and 3′ end sequences of the open circle probe.

[0289] The open circle probe can also be inactivated by formation of the intramolecular stem structure during the amplification reaction. As long as the end remains in the intramolecular stem structure, it is not available for priming nucleic acid synthesis. This form of inactivation is aided by design the intramolecular stem structure, or selecting amplification conditions, such that the intramolecular hybrid remains stable during rolling circle amplification.

[0290] One form of the disclosed open circle probes includes a loop as part of the intramolecular stem structure. It is preferred that the loop contain sequences complementary to the target sequence. This allows the loop to nucleate hybridization of the open probe to the target sequence. Preferred forms of the loop-containing probes are characterized by a sequence discrimination capability that is markedly better that the comparable linear probes due to the competition between the structural interferences between folding due to intramolecular stem formation and linear rigidity due to hybridization of the probe sequence to the target (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). Preferred open circle probes of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end(s) of the open circle probe involved in an intramolecular stem structure unavailable for ligation to circularize the probe and leave the 3′ end available for inactivating extension. The presence of target sequence causes the correctly matched open circle probe to unfold, allowing the ends to hybridize to the target sequence and be coupled (see FIG. 3). Where sequences in the loop nucleate hybridization of the open circle probe to a target sequence, loop hybridization to a non-target sequence is unlikely to lead to circularization of the open circle probe. This is because it is unlikely that a non-target sequence will include adjacent sequences to which both the loop and open circle probe end can hybridize (see FIG. 4).

[0291] A hybridization nucleating loop can also be used in linear primers used for nucleic acid replication and amplification. Such a primer forms an intramolecular stem structure, including a loop. Loop-containing primers of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end of the primer involved in an intramolecular stem structure unavailable for priming. The legitimate primer complement sequence causes the correctly matched primer to unfold, allowing the end to hybridize to the primer complement sequence and prime synthesis. Where sequences in the loop nucleate hybridization of the primer, loop hybridization to an illegitimate sequence is unlikely to lead to priming. This is because it is unlikely that an illegitimate sequence will include adjacent sequences to which both the loop and the primer end can hybridize. Useful primers forming intramolecular stem structures can be fluorescent change primers. For example, including proximity-sensitive labels used in molecular beacon probes in such primers allows hybridization and priming by the primers to be detected through activation of the label upon disruption of the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)).

[0292] Some forms of the disclosed compositions and method can involve or produce a combination of the above effects. That is, nucleic acid amplification reactions can combine two or more of reduction, prevention, or elimination of artifacts; increased efficiency; increased specificity; and/or increased consistency. The disclosed method can combine, for example, the use of open circle probes that can form intramolecular stem structures; the use of matched open circle probe sets in the same amplification reaction; the use of detection primers and detection during the amplification reaction; the use of a plurality of detection rolling circle replication primer, a secondary DNA strand displacement primer and a common rolling circle replication primer in the same amplification reaction; and/or the use of peptide nucleic acid quenchers associated with detection rolling circle replication primers. Such combinations can produce, in the same amplification reaction, the benefits of each of the combined components.

[0293] The disclosed method is useful for detection, quantitation, and/or location of any desired analyte. The disclosed method can be multiplexed to detect numerous different analytes simultaneously or used in a single assay. Thus, the disclosed method is useful for detecting, assessing, quantitating, profiling, and/or cataloging gene expression and the presence of protein in biological samples. The disclosed method is also particularly useful for detecting and discriminating single nucleotide differences in nucleic acid sequences. This specificity is possible due to the sensitivity of the intramolecular stem structure in loop-containing probes and primers to mismatches between the loop sequence and a prospective target sequence. Thus, the disclosed method is useful for extensive multiplexing of target sequences for sensitive and specific detection of the target sequences themselves or analytes to which the target sequences have been associated. The disclosed method is also useful for detecting, assessing, quantitating, and/or cataloging single nucleotide polymorphisms, and other sequence differences between nucleic acids, nucleic acid samples, and sources of nucleic acid samples.

[0294] The disclosed method is useful for detecting any desired sequence or other analyte, such as proteins and peptides. In particular, the disclosed method can be used to localize or amplify signal from any desired analyte. For example, the disclosed method can be used to assay tissue, transgenic cells, bacterial or yeast colonies, cellular material (for example, whole cells, proteins, DNA fibers, interphase nuclei, or metaphase chromosomes on slides, arrayed genomic DNA, RNA), and samples and extracts from any of biological source. Where target sequences are associated with an analyte, different target sequences, and thus different analytes, can be sensitively distinguished. Specificity of such detection is aided by sensitivity of a loop in an open circle probe to mismatches.

[0295] The disclosed method is applicable to numerous areas including, but not limited to, analysis of proteins present in a sample (for example, proteomics analysis), disease detection, mutation detection, protein expression profiling, RNA expression profiling, gene discovery, gene mapping (molecular haplotyping), agricultural research, and virus detection. Preferred uses include protein and peptide detection in situ in cells, on microarrays, protein expression profiling; mutation detection; detection of abnormal proteins or peptides (for example, overexpression of an oncogene protein or absence of expression of a tumor suppressor protein); expression in cancer cells; detection of viral proteins in cells; viral protein expression; detection of inherited diseases such as cystic fibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia; assessment of predisposition for cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, testicular cancer, pancreatic cancer. The disclosed method can also be used for detection of nucleic acids in situ in cells, on microarrays, on DNA fibers, and on genomic DNA arrays; detection of RNA in cells; RNA expression profiling; molecular haplotyping; mutation detection; detection of abnormal RNA (for example, overexpression of an oncogene or absence of expression of a tumor suppressor gene); expression in cancer cells; detection of viral genome in cells; viral RNA expression; detection of inherited diseases such as cystic fibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia; assessment of predisposition for cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, testicular cancer, pancreatic cancer.

[0296] A. The Ligation Operation

[0297] An open circle probe, optionally in the presence of one or more gap oligonucleotides, can be incubated with a sample containing nucleic acids, under suitable hybridization conditions, and then ligated to form a covalently closed circle. The ligated open circle probe is a form of amplification target circle. This operation is similar to ligation of padlock probes described by Nilsson et al., Science, 265:2085-2088 (1994). The ligation operation allows subsequent amplification to be dependent on the presence of a target sequence. Suitable ligases for the ligation operation are described above. Ligation conditions are generally known. Most ligases require-Mg++. There are two main types of ligases, those that are ATP-dependent and those that are NAD-dependent. ATP or NAD, depending on the type of ligase, should be present during ligation.

[0298] The disclosed hairpin open circle probes reduce the incidence of non-specific ligation of open circle probe ends because one or both of the ends remain in the intramolecular stem structure unless hybridized to a target sequence. Loop-containing open circle probes allow better discrimination of target sequence hybridization by the open circle probes. As discussed below, hybridization of sequences in the loop to target sequence can disrupt the intramolecular stem structure. In the absence of target sequence, the stem structure remains intact. The disclosed open circle probes are particularly suited for use in ligation reactions with multiple different open circle probes and a complex nucleic acid sample.

[0299] The target sequence for an open circle probe can be any nucleic acid or other compound to which the target probe portions of the open circle probe can hybridize in the proper alignment. Target sequences can be found in any nucleic acid molecule from any nucleic acid sample. Thus, target sequences can be in nucleic acids in cell or tissue samples, reactions, and assays. Target sequences can also be artificial nucleic acids (or other compounds to which the target probe portions of the open circle probe can hybridize in the proper alignment). For example, nucleic acid tags can be associated with various of the disclosed compounds to be detected using open circle probes. Thus, a reporter binding agent can contain a target sequence to which an open circle probe can hybridize. In these cases, the target sequence provides a link between the target molecule being detected and the amplification of signal mediated by the open circle probe. When matched open circle probe sets are used, the target sequences will be related based on the relationship of the open circle probes in the set.

[0300] When RNA is to be detected, it is preferred that a reverse transcription operation be performed to make a DNA target sequence. Alternatively, an RNA target sequence can be detected directly by using a ligase that can perform ligation on a DNA:RNA hybrid substrate. A preferred ligase for this is T4 DNA ligase.

[0301] B. The Amplification Operation

[0302] The basic form of amplification operation is rolling circle replication of a circular DNA molecule (that is, a circularized open circle probe or an amplification target circle). The circular open circle probes formed by specific ligation and amplification target circles serve as substrates for a rolling circle replication. This reaction requires two reagents: (a) a rolling circle replication primer, which is complementary to the primer complement portion of the OCP or ATC, and (b) a rolling circle DNA polymerase. The DNA polymerase catalyzes primer extension and strand displacement in a processive rolling circle polymerization reaction that proceeds as long as desired, generating a molecule of 100,000 nucleotides or more that contains up to approximately 1000 tandem copies or more of a sequence complementary to the amplification target circle or open circle probe. This tandem sequence DNA (TS-DNA) consists of, in the case of OCPs, alternating target sequence and spacer sequence. Note that the spacer sequence of the TS-DNA is the complement of the sequence between the left target probe and the right target probe in the original open circle probe. Some forms of the disclosed method use two types of rolling circle replication primer (detection rolling circle replication primers and common rolling circle replication primers) and secondary DNA strand displacement primers in the amplification reaction. The use of these different primers provide benefits in the amplification operation as described elsewhere herein.

[0303] Detection of amplification during the amplification operation (that is, real-time detection) is desirable. This can be accomplished in any suitable manner. A particularly useful means of obtaining real-time detection is the use of fluorescent change probes and/or primers in the amplification operation. With suitably designed fluorescent change probes and primers, fluorescent signals can be generated as amplification proceeds. In most such cases, the fluorescent signals will be in proportion to the amount of amplification product and/or amount of target sequence or target molecule.

[0304] During rolling circle replication one may additionally include radioactive, or modified nucleotides such as bromodeoxyuridine triphosphate, in order to label the DNA generated in the reaction. Alternatively, one may include suitable precursors that provide a binding moiety such as biotinylated nucleotides (Langer et al. (1981)). Unmodified TS-DNA can be detected using any nucleic acid detection technique.

[0305] As well as rolling circle replication, the amplification operation can include additional nucleic acid replication or amplification processes. For example, TS-DNA can itself be replicated to form secondary TS-DNA. This process is referred to as secondary DNA strand displacement. The combination of rolling circle replication and secondary DNA strand displacement is referred to as linear rolling circle amplification (LRCA). The secondary TS-DNA can itself be replicated to form tertiary TS-DNA in a process referred to as tertiary DNA strand displacement. Secondary and tertiary DNA strand displacement can be performed sequentially or simultaneously. When performed simultaneously, the result is strand displacement cascade amplification. The combination of rolling circle replication and strand displacement cascade amplification is referred to as exponential rolling circle amplification (ERCA). Secondary TS-DNA, tertiary TS-DNA, or both can be amplified by transcription. Exponential rolling circle amplification is a preferred form of amplification operation. Particularly useful is ERCA mediated by the use of detection rolling circle replication primers, common rolling circle replication primers and secondary DNA strand displacement primers in the amplification reaction.

[0306] After RCA, a round of LM-RCA can be performed on the TS-DNA produced in the first RCA. This new round of LM-RCA is performed with a new open circle probe, referred to as a secondary open circle probe, having target probe portions complementary to a target sequence in the TS-DNA produced in the first round. When such new rounds of LM-RCA are performed, the amplification is referred to as nested LM-RCA. Nested LM-RCA can also be performed on ligated OCPs or ATCs that have not been amplified. In this case, LM-RCA can be carried out using either ATCs or target-dependent ligated OCPs. This is especially useful for in situ detection. For in situ detection, the first, unamplified OCP, which is topologically locked to its target sequence, can be subjected to nested LM-RCA. By not amplifying the first OCP, it can remain hybridized to the target sequence while LM-RCA amplifies a secondary OCP topologically locked to the first OCP. Nested LM-RCA is described in U.S. Pat. No. 6,143,495.

[0307] C. Extension

[0308] The disclosed method can use probes and primers that form intramolecular stem structures to reduce or eliminate non-specific and other undesired nucleic acid replication. This is accomplished by virtue of the probe and primer design (as described elsewhere herein) and results in “inactivation” of the probes and primer if they are not involved in legitimate hybrid. Such inactivation refers to the reduced ability of the probe or primer to hybridize to sequences other than their intended target sequence. As used herein, inactivation of probes and primers does not require complete loss of-non-specific hybridization; reduction in non-specific hybridization is sufficient.

[0309] The disclosed open circle probes that can form intramolecular stem structures can be inactivated in several ways. For example, where the 3′ end of an open circle probe is involved in an intramolecular stem structure, the 3′ end can be extended in a replication reaction using the open circle probe sequences as template (see FIG. 2B). The result is stabilization of the intramolecular stem structure and a change in the 3′ end sequence. Stabilization of the stem structure results in a reduction or elimination of the ability of the open circle probe to prime nucleic acid synthesis because the 3′ end is stably hybridized to sequences in the open circle probe under the conditions used for nucleic acid replication. Change in the sequence of the 3′ end can reduce of the ability of the open circle probe to prime nucleic acid synthesis because the changed 3′ sequences may not be as closely related to sequences involved in the amplification reaction or assay. Change in the sequence of the 3′ end can reduce of the ability of the open circle probe to serve as a template for rolling circle amplification. For example, even if the open circle probe with extended 3′ end were circularized, the rolling circle replication primer could be prevented from priming replication of such a circle if the primer complement sequence on the open circle probe were interrupted by the added sequences. This can be accomplished by, for example, designing the open circle probe to have the primer complement sequence include both 5′ and 3′ end sequences of the open circle probe.

[0310] D. Sequestration

[0311] The disclosed open circle probes that can form intramolecular stem structures can also be inactivated by formation of the intramolecular stem structure during the amplification reaction. As long as the end remains in the intramolecular stem structure (that is, as long as it is sequestered in the stem structure), it is not available for priming nucleic acid synthesis. This form of inactivation is aided by design the intramolecular stem structure, or selecting amplification conditions, such that the intramolecular hybrid remains stable during rolling circle amplification. Extension of the end as described above also results in sequestration of the end in the intramolecular stem structure.

[0312] Discrimination of rolling circle replication primer hybridization also can be accomplished by hybridizing primer to primer complement portions of OCPs or ATCs under conditions that favor only exact sequence matches leaving other rolling circle replication primers unhybridized. The unhybridized rolling circle replication primers will retain or re-form the intramolecular hybrid. Discrimination of DNA strand displacement primer hybridization can be accomplished in a similar manner by hybridizing primer to TS-DNA under conditions that favor only exact sequence matches leaving other DNA strand displacement primers unhybridized.

[0313] E. Loop Hybridization Disruption

[0314] One form of the disclosed open circle probes includes a loop as part of the intramolecular stem structure. It is preferred that the loop contain sequences complementary to the target sequence. This allows the loop to nucleate hybridization of the open probe to the target sequence. Preferred forms of the loop-containing probes are characterized by a sequence discrimination capability that is markedly better that the comparable linear probes due to the competition between the structural interferences between folding due to intramolecular stem formation and linear rigidity due to hybridization of the probe sequence to the target (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)). Preferred open circle probes of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end(s) of the open circle probe involved in an intramolecular stem structure unavailable for ligation to circularize the probe and leave the 3′ end available for inactivating extension. The presence of target sequence causes the correctly matched open circle probe to unfold, allowing the ends to hybridize to the target sequence and be coupled (see FIG. 3). Where sequences in the loop nucleate hybridization of the open circle probe to a target sequence, loop hybridization to a non-target sequence is unlikely to lead to circularization of the open circle probe. This is because it is unlikely that a non-target sequence will include adjacent sequences to which both the loop and open circle probe end can hybridize (see FIG. 4).

[0315] A hybridization nucleating loop can also be used in linear primers used for nucleic acid replication and amplification. Such a primer forms an intramolecular stem structure, including a loop. Loop-containing primers of this type will not hybridize to mismatched sequences under suitable conditions because duplex hybridization of probe to target does not effectively compete with intramolecular stem formation of the structured probe. This makes the end of the primer involved in an intramolecular stem structure unavailable for priming. The legitimate primer complement sequence causes the correctly matched primer to unfold, allowing the end to hybridize to the primer complement sequence and prime synthesis. Where sequences in the loop nucleate hybridization of the primer, loop hybridization to an illegitimate sequence is unlikely to lead to priming. This is because it is unlikely that an illegitimate sequence will include adjacent sequences to which both the loop and the primer end can hybridize. Useful primers forming intramolecular stem structures can be fluorescent change primers. For example, including proximity-sensitive labels used in molecular beacon probes in such primers allows hybridization and priming by the primers to be detected through activation of the label upon disruption of the intramolecular stem structure (Tyagi and Kramer, Nat Biotechnol 14(3):303-8 (1996); Bonnet et al., Proc Natl Acad Sci U S A 96(11):6171-6 (1999)).

[0316] F. DNA Strand Displacement

[0317] DNA strand displacement is one way to amplify TS-DNA. Secondary DNA strand displacement is accomplished by hybridizing secondary DNA strand displacement primers to TS-DNA and allowing a DNA polymerase to synthesize DNA from these primed sites (see FIG. 11 in U.S. Pat. No. 6,143,495). Because a complement of the secondary DNA strand displacement primer occurs in each repeat of the TS-DNA, secondary DNA strand displacement can result in a high level of amplification. The product of secondary DNA strand displacement is referred to as secondary tandem sequence DNA or TS-DNA-2. Secondary DNA strand displacement can be accomplished by performing RCA to produce TS-DNA, mixing secondary DNA strand displacement primer with the TS-DNA, and incubating under conditions promoting replication of the tandem sequence DNA. The disclosed hairpin open circle probes are especially useful for DNA strand displacement because inactivated hairpin open circle probes will not compete with secondary DNA strand displacement primers for hybridization to TS-DNA. The DNA strand displacement primers are preferably hairpin DNA strand displacement primers.

[0318] Secondary DNA strand displacement can also be carried out simultaneously with rolling circle replication. This is accomplished by mixing secondary DNA strand displacement primer with the reaction prior to rolling circle replication. As a secondary DNA strand displacement primer is elongated, the DNA polymerase will run into the 5′ end of the next hybridized secondary DNA strand displacement molecule and will displace its 5′ end. In this fashion a tandem queue of elongating DNA polymerases is formed on the TS-DNA template. As long as the rolling circle reaction continues, new secondary DNA strand displacement primers and new DNA polymerases are added to TS-DNA at the growing end of the rolling circle. The generation of TS-DNA-2 and its release into solution by strand displacement is shown diagrammatically in FIG. 11 in U.S. Pat. No. 6,143,495. For simultaneous rolling circle replication and secondary DNA strand displacement, it is preferred that the rolling circle DNA polymerase be used for both replications. This allows optimum conditions to be used and results in displacement of other strands being synthesized downstream. Secondary DNA strand displacement can follow any DNA replication operation, such as RCA, LM-RCA or nested LM-RCA.

[0319] Generally, secondary DNA strand displacement can be performed by, simultaneous with or following RCA, mixing a secondary DNA strand displacement primer with the reaction mixture and incubating under conditions that promote both hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, and replication of the tandem sequence DNA, where replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA.

[0320] When secondary DNA strand displacement is carried out in the presence of a tertiary DNA strand displacement primer (or an equivalent primer), an exponential amplification of TS-DNA sequences takes place. This special and preferred mode of DNA strand displacement is referred to as strand displacement cascade amplification (SDCA) and is a form of exponential rolling circle amplification (ERCA). In SDCA, a secondary DNA strand displacement primer primes replication of TS-DNA to form TS-DNA-2, as described above. The tertiary DNA strand displacement primer strand can then hybridize to, and prime replication of, TS-DNA-2 to form TS-DNA-3. Strand displacement of TS-DNA-3 by the adjacent, growing TS-DNA-3 strands makes TS-DNA-3 available for hybridization with secondary DNA strand displacement primer. This results in another round of replication resulting in TS-DNA-4 (which is equivalent to TS-DNA-2). TS-DNA-4, in turn, becomes a template for DNA replication primed by tertiary DNA strand displacement primer. The cascade continues this manner until the reaction stops or reagents become limiting. This reaction amplifies DNA at an almost exponential rate. In a useful mode of SDCA, the rolling circle replication primers serve as the tertiary DNA strand displacement primer, thus eliminating the need for a separate primer.

[0321] For this mode, the rolling circle replication primer should be used at a concentration sufficiently high to obtain rapid priming on the growing TS-DNA-2 strands. To optimize the efficiency of SDCA, it is preferred that a sufficient concentration of secondary DNA strand displacement primer and tertiary DNA strand displacement primer be used to obtain sufficiently rapid priming of the growing TS-DNA strand to out compete TS-DNA for binding to its complementary TS-DNA. Optimization of primer concentrations are described in U.S. Pat. No. 6,143,495 and can be aided by analysis of hybridization kinetics (Young and Anderson, “Quantitative analysis of solution hybridization” in Nucleic Acid Hybridization: A Practical Approach (IRL Press, 1985) pages 47-71).

[0322] A useful form of strand displacement cascade amplification uses one or more detection rolling circle replication primers, one or more common rolling circle replication primers, and one or more secondary DNA strand displacement primers. Either or both of the rolling circle replication primers can function as a tertiary DNA strand displacement primer (they will also function as rolling circle replication primers).

[0323] Generally, strand displacement cascade amplification can be performed by, simultaneous with, or following, RCA, mixing a secondary DNA strand displacement primer and a tertiary DNA strand displacement primer with the reaction mixture and incubating under conditions that promote hybridization between the tandem sequence DNA and the secondary DNA strand displacement primer, replication of the tandem sequence DNA—where replication of the tandem sequence DNA results in the formation of secondary tandem sequence DNA hybridization between the secondary tandem sequence DNA and the tertiary DNA strand displacement primer, and replication of secondary tandem sequence DNA—where replication of the secondary tandem sequence DNA results in formation of tertiary tandem sequence DNA (TS-DNA-3).

[0324] Secondary and tertiary DNA strand displacement can also be carried out sequentially. Following a first round of secondary DNA strand displacement, a tertiary DNA strand displacement primer can be mixed with the secondary tandem sequence DNA and incubated under conditions that promote hybridization between the secondary tandem sequence DNA and the tertiary DNA strand displacement primer, and replication of secondary tandem sequence DNA, where replication of the secondary tandem sequence DNA results in formation of tertiary tandem sequence DNA (TS-DNA-3). This round of strand displacement replication can be referred to as tertiary DNA strand displacement. However, all rounds of strand displacement replication following rolling circle replication can also be referred to collectively as DNA strand displacement or secondary DNA strand displacement.

[0325] A modified form of secondary DNA strand displacement results in amplification of TS-DNA and is referred to as opposite strand amplification (OSA). OSA is the same as secondary DNA strand displacement except that a special form of rolling circle replication primer is used that prevents it from hybridizing to TS-DNA-2. Opposite strand amplification is described in U.S. Pat. No. 6,143,495.

[0326] The DNA generated by DNA strand displacement can be labeled and/or detected using the same labels, labeling methods, and detection methods described for use with TS-DNA. Most of these labels and methods are adaptable for use with nucleic acids in general. A preferred method of labeling the DNA is by incorporation of labeled nucleotides during synthesis.

[0327] G. Detection of Amplification Products

[0328] Products of the amplification operation can be detected using any nucleic acid detection technique. Many techniques are known for detecting nucleic acids. Several preferred forms of detection are described below. The nucleotide sequence of the amplified sequences also can be determined using any suitable technique. Particularly useful are techniques for real-time detection of amplification. Fluorescent change probes and primers are useful for detection in general and real-time detection in particular.

[0329] 1. Primary Labeling

[0330] Primary labeling consists of incorporating labeled moieties, such as fluorescent nucleotides, biotinylated nucleotides, digoxygenin-containing nucleotides, or bromodeoxyuridine, during rolling circle replication in RCA, or during transcription in RCT. For example, one may incorporate cyanine dye UTP analogs (Yu et al. (1994)) at a frequency of 4 analogs for every 100 nucleotides. A preferred method for detecting nucleic acid amplified in situ is to label the DNA during amplification with BrdUrd, followed by binding of the incorporated BUDR with a biotinylated anti-BUDR antibody (Zymed Labs, San Francisco, Calif.), followed by binding of the biotin moieties with Streptavidin-Peroxidase (Life Sciences, Inc.), and finally development of fluorescence with Fluorescein-tyramide (DuPont de Nemours & Co., Medical Products Dept.).

[0331] A useful form of primary labeling is the use of fluorescent change primers in the amplification operation. Fluorescent change primers exhibit a change in fluorescence intensity or wavelength based on a change in the form of conformation of the primer and the amplified nucleic acid. Stem quenched primers are primers that when not hybridized to a complementary sequence form a stem structure (either an intramolecular stem structure or an intermolecular stem structure) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. In the disclosed method, stem quenched primers are used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of stem quenched primers are peptide nucleic acid quenched primers and hairpin quenched primers.

[0332] Peptide nucleic acid quenched primers are primers associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor to form a stem structure. The primer contains a fluorescent label or a quenching moiety and is associated with either a peptide nucleic acid quencher or a peptide nucleic acid fluor, respectively. This puts the fluorescent label in proximity to the quenching moiety. When the primer is replicated, the peptide nucleic acid is displaced, thus allowing the fluorescent label to produce a fluorescent signal.

[0333] Hairpin quenched primers are primers that when not hybridized to a complementary sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Hairpin quenched primers are typically used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of hairpin quenched primers are Amplifluor primers and scorpion primers.

[0334] 2. Secondary Labeling

[0335] Secondary labeling consists of using suitable molecular probes, such as detection probes, to detect the amplified nucleic acids. For example, an open circle may be designed to contain several repeats of a known arbitrary sequence, referred to as detection tags. A secondary hybridization step can be used to bind detection probes to these detection tags (see FIG. 7 in U.S. Pat. No. 6,143,495). The detection probes may be labeled as described above with, for example, an enzyme, fluorescent moieties, or radioactive isotopes. By using three detection tags per open circle probe, and four fluorescent moieties per each detection probe, one may obtain a total of twelve fluorescent signals for every open circle probe repeat in the TS-DNA, yielding a total of 12,000 fluorescent moieties for every ligated open circle probe that is amplified by RCA.

[0336] A useful form of secondary labeling is the use of fluorescent change probes and primers in or following the amplification operation. Hairpin quenched probes are probes that when not bound to a target sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the probe binds to a target sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Examples of hairpin quenched probes are molecular beacons, fluorescent triplex oligos, and QPNA probes.

[0337] Cleavage activated probes are probes where fluorescence is increased by cleavage of the probe. Cleavage activated probes can include a fluorescent label and a quenching moiety in proximity such that fluorescence from the label is quenched. When the probe is clipped or digested (typically by the 5′-3′ exonuclease activity of a polymerase during or following amplification), the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. TaqMan probes are an example of cleavage activated probes.

[0338] Cleavage quenched probes are probes where fluorescence is decreased or altered by cleavage of the probe. Cleavage quenched probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity, fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. The probes are thus fluorescent, for example, when hybridized to a target sequence. When the probe is clipped or digested (typically by the 5′-3′ exonuclease activity of a polymerase during or after amplification), the donor moiety is no longer in proximity to the acceptor fluorescent label and fluorescence from the acceptor decreases. If the donor moiety is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor. The overall effect would then be a reduction of acceptor fluorescence and an increase in donor fluorescence. Donor fluorescence in the case of cleavage quenched probes is equivalent to fluorescence generated by cleavage activated probes with the acceptor being the quenching moiety and the donor being the fluorescent label. Cleavable FRET (fluorescence resonance energy transfer) probes are an example of cleavage quenched probes.

[0339] Fluorescent activated probes are probes or pairs of probes where fluorescence is increased or altered by hybridization of the probe to a target sequence. Fluorescent activated probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity (when the probes are hybridized to a target sequence), fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. Fluorescent activated probes are typically pairs of probes designed to hybridize to adjacent sequences such that the acceptor and donor are brought into proximity. Fluorescent activated probes can also be single probes containing both a donor and acceptor where, when the probe is not hybridized to a target sequence, the donor and acceptor are not in proximity but where the donor and acceptor are brought into proximity when the probe hybridized to a target sequence. This can be accomplished, for example, by placing the donor and acceptor on opposite ends a the probe and placing target complement sequences at each end of the probe where the target complement sequences are complementary to adjacent sequences in a target sequence. If the donor moiety of a fluorescent activated probe is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor (that is, when the probes are not hybridized to the target sequence). When the probes hybridize to a target sequence, the overall effect would then be a reduction of donor fluorescence and an increase in acceptor fluorescence. FRET probes are an example of fluorescent activated probes. Stem quenched primers (such as peptide nucleic acid quenched primers and hairpin quenched primers) can be used as secondary labels.

[0340] 3. Multiplexing and Hybridization Array Detection

[0341] RCA is easily multiplexed by using sets of different open circle probes, each open circle probe carrying different target probe sequences designed for binding to unique targets and, optionally, each open circle probe having a different detection primer complement portion corresponding to different detection rolling circle replication primers. Note that although the target probe sequences designed for each target and the detection primer complement portions are different, the common primer complement portions and secondary DNA strand displacement primer matching portions may remain the same for all of the open circle probes, and thus some of the primers for rolling circle amplification can remain the same for all targets. Only those open circle probes that are able to find their targets will give rise to TS-DNA. Use of different fluorescent labels with different detection rolling circle replication primers allows specific detection of different open circle probes (and thus, of different targets).

[0342] The TS-DNA molecules generated by RCA are of high molecular weight and low complexity; the complexity being the length of the open circle probe. There are two alternatives for capturing a given TS-DNA to a fixed position in a solid-state detector. One is to include within the spacer region of the open circle probes a unique address tag sequence for each unique open circle probe. TS-DNA generated from a given open circle probe will then contain sequences corresponding to a specific address tag sequence. A second and preferred alternative is to use the target sequence present on the TS-DNA as the address tag.

[0343] 4. Combinatorial Multicolor Coding

[0344] One form of multiplex detection involves the use of a combination of labels that either fluoresce at different wavelengths or are colored differently. One of the advantages of fluorescence for the detection of hybridization probes is that several targets can be visualized simultaneously in the same sample. Using a combinatorial strategy, many more targets can be discriminated than the number of spectrally resolvable fluorophores. Combinatorial labeling provides the simplest way to label probes in a multiplex fashion since a probe fluor is either completely absent (−) or present in unit amounts (+); image analysis is thus more amenable to automation, and a number of experimental artifacts, such as differential photobleaching of the fluors and the effects of changing excitation source power spectrum, arc avoided.

[0345] The combinations of labels establish a code for identifying different detection probes and, by extension, different target molecules to which those detection probes are associated with. This labeling scheme is referred to as Combinatorial Multicolor Coding (CMC). Such coding is described by Speicher et al., Nature Genetics 12:368-375 (1996). Use of CMC in connection with rolling circle amplification is described in U.S. Pat. No. 6,143,495. Any number of labels, which when combined can be separately detected, can be used for combinatorial multicolor coding. It is preferred that 2, 3, 4, 5, or 6 labels be used in combination. It is most preferred that 6 labels be used. The number of labels used establishes the number of unique label combinations that can be formed according to the formula 2N−1, where N is the number of labels. According to this formula, 2 labels forms three label combinations, 3 labels forms seven label combinations, 4 labels forms 15 label combinations, 5 labels form 31 label combinations, and 6 labels forms 63 label combinations.

[0346] For combinatorial multicolor coding, a group of different detection probes are used as a set. Each type of detection probe in the set is labeled with a specific and unique combination of fluorescent labels. For those detection probes assigned multiple labels, the labeling can be accomplished by labeling each detection probe molecule with all of the required labels. Alternatively, pools of detection probes of a given type can each be labeled with one of the required labels. By combining the pools, the detection probes will, as a group, contain the combination of labels required for that type of detection probe. Where each detection probe is labeled with a single label, label combinations can also be generated by using OCPs or ATCs with coded combinations of detection tags complementary to the different detection probes. In this scheme, the OCPs or ATCs will contain a combination of detection tags representing the combination of labels required for a specific label code. Further illustrations are described in U.S. Pat. No. 6,143,495.

[0347] Rolling circle amplification can be engineered to produce TS-DNA of different lengths in an assay involving multiple ligated OCPs or ATCs. The resulting TS-DNA of different length can be distinguished simply on the basis of the size of the detection signal they generate. Thus, the same set of detection probes could be used to distinguish two different sets of generated TS-DNA. In this scheme, two different TS-DNAs, each of a different size but assigned the same color code, would be distinguished by the size of the signal produced by the hybridized detection probes. In this way, a total of 126 different targets can be distinguished on a single solid-state sample using a code with 63 combinations, since the signals will come in two flavors, low amplitude and high amplitude. Thus one could, for example, use the low amplitude signal set of 63 probes for detection of an oncogene mutations, and the high amplitude signal set of 63 probes for the detection of a tumor suppressor p53 mutations.

[0348] Speicher et al. describes a set of fluors and corresponding optical filters spaced across the spectral interval 350-770 nm that give a high degree of discrimination between all possible fluor pairs. This fluor set, which is preferred for combinatorial multicolor coding, consists of 4′-6-diamidino-2-phenylinodole (DAPI), fluorescein (FITC), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Any subset of this preferred set can also be used where fewer combinations are required. The absorption and emission maxima, respectively, for these fluors are: DAPI (350 nm; 456 nm), FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm). The excitation and emission spectra, extinction coefficients and quantum yield of these fluors are described by Ernst et al., Cytometry 10:3-10 (1989), Mujumdar et al., Cytometry 10:11-19 (1989), Yu, Nucleic Acids Res. 22:3226-3232 (1994), and Waggoner, Meth. Enzymology 246:362-373 (1995). These fluors can all be excited with a 75W Xenon arc.

[0349] To attain selectivity, filters with bandwidths in the range of 5 to 16 nm are preferred. To increase signal discrimination, the fluors can be both excited and detected at wavelengths far from their spectral maxima. Emission bandwidths can be made as wide as possible. For low-noise detectors, such as cooled CCD cameras, restricting the excitation bandwidth has little effect on attainable signal to noise ratios. A list of preferred filters for use with the preferred fluor set is listed in Table 1 of Speicher et al. It is important to prevent infra-red light emitted by the arc lamp from reaching the detector; CCD chips are extremely sensitive in this region. For this purpose, appropriate IR blocking filters can be inserted in the image path immediately in front of the CCD window to minimize loss of image quality. Image analysis software can then be used to count and analyze the spectral signatures of fluorescent dots.

[0350] Discrimination of individual signals in combinatorial multicolor coding can be enhanced by collapsing TS-DNA generated during amplification. As described elsewhere herein, this is preferably accomplished using collapsing detection probes, biotin-antibody conjugates, or a combination of both. A collapsed TS-DNA can occupy a space of no more than 0.3 microns in diameter. Based on this, it is expected that up to a million discrete signals can be detected in a 2.5 mm sample dot. Such discrimination also results in a large dynamic range for quantitative signal detection. Thus, the relative numbers of different types of signals (such as multicolor codes) can be determined over a wide range. This is expected to allow determination of, for example, the relative amount of different target molecules, such as proteins, in a sample. Such comparative detections would be useful in, for example, proteomics analyses of cell and tissue samples. This would also allow determination of whether a particular target sequence is homozygous or heterozygous in a genomic DNA sample, whether a target sequence was inherited or represents a somatic mutation, and the genetic heterogeneity of a genomic DNA sample, such as a tumor sample.

[0351] 5. Detecting Multiple Target Sequences

[0352] Multiplex RCA assays are useful for detecting multiple proteins. A single LM-RCA assay can be used to detect the presence of one or more members of a group of any number of target sequences. By associating different target sequences with different proteins (using reporter binding agents specific for the proteins of interest), each different protein can be detected by differential detection of the various target sequences. This can be accomplished, for example, by designing an open circle probe (and associated gap oligonucleotides, if desired) for each target sequence in the group, where the target probe portions and the detection primer complement portions of each open circle probe are different but the sequence of the common primer complement portions and secondary DNA strand displacement matching portions of all the open circle probes are the same. All of the open circle probes are placed in the same OCP-target sample mixture, and the same primers are used to amplify. For each target sequence present in the assay (those associated with proteins present in the target sample, for example), the OCP for that target will be ligated into a circle and the circle will be amplified to form TS-DNA. Since the detection primer complement portions are different, amplification of the different OCPs can be detected (using, for example, detection rolling circle replication primers that are fluorescent change primers). Alternatively, the open circle probes can each target a different target sequence in the group, where the target probe portions and the sequence of the detection tag portions of each open circle probe are different but the sequence of the primer portions of all the open circle probes are the same. Different detection probes are used to detect the various TS-DNAs (each having specific detection tag sequences). For each target sequence present in the assay (those associated with proteins present in the target sample, for example), the OCP for that target will be ligated into a circle and the circle will be amplified to form TS-DNA. Since the detection tags on TS-DNA resulting from amplification of the OCPs are the different, TS-DNA resulting from ligation each OCP can be detected individually in that assay.

[0353] 6. Detecting Groups of Target Sequences

[0354] Multiplex RCA assays are particularly useful for detecting any of a set of target sequences in a defined group. For example, the disclosed method can be used to detect mutations in genes where numerous distinct mutations are associated with certain diseases or where mutations in multiple genes are involved. For example, although the gene responsible for Huntington's chorea has been identified, a wide range of mutations in different parts of the gene occur among affected individuals. The result is that no single test has been devised to detect whether an individual has one or more of the many Huntington's mutations. A single LM-RCA assay can be used to detect the presence of one or more members of a group of any number of target sequences. This can be accomplished, for example, by designing an open circle probe (and associated gap oligonucleotides, if desired) for each target sequence in the group, where the target probe portions of each open circle probe are different but the sequence of the primer complement portions and secondary DNA strand displacement primer matching portions of all the open circle probes are the same. All of the open circle probes are placed in the same OCP-target sample mixture, and the same primers are used to amplify and detect TS-DNA. If any of the target sequences are present in the target sample, the OCP for that target will be ligated into a circle and the circle will be amplified to form TS-DNA. Since the detection rolling circle replication primers (preferably using fluorescent change primers) for all of the OCPs are the same, TS-DNA resulting from ligation of any of the OCPs will be detected in that assay. Detection indicates that at least one member of the target sequence group is present in the target sample. This allows detection of a trait associated with multiple target sequences in a single tube or well.

[0355] If a positive result is found, the specific target sequence involved can be identified by using a multiplex assay. This can be facilitated by including different detection tags in each of the OCPs of the group. In this way, TS-DNA generated from each different OCP, representing each different target sequence, can be individually detected. It is convenient that such multiple assays need be performed only when an initial positive result is found.

[0356] The above scheme can also be used with arbitrarily chosen groups of target sequences in order to screen for a large number of target sequences without having to perform an equally large number of assays. Initial assays can be performed as described above, each using a different group of OCPs designed to hybridize to a different group of target sequences. Additional assays to determine which target sequence is present can then be performed on only those groups that produce TS-DNA. Such group assays can be further nested if desired.

[0357] Multiplex detection can also be accomplished by designing an open circle probe (and associated gap oligonucleotides, if desired) for each target sequence in the group, where the target probe portions of each open circle probe are different but the sequence of the primer portions and the sequence of the detection tag portions of all the open circle probes are the same. All of the open circle probes are placed in the same OCP-target sample mixture, and the same primer and detection probe are used to amplify and detect TS-DNA. If any of the target sequences are present in the target sample, the OCP for that target will be ligated into a circle and the circle will be amplified to form TS-DNA. Since the detection tags on TS-DNA resulting from amplification of any of the OCPs are the same, TS-DNA resulting from ligation of any of the OCPs will be detected in that assay. Detection indicates that at least one member of the target sequence group is present in the target sample. This allows detection of a trait associated with multiple target sequences in a single tube or well.

[0358] If a positive result is found, the specific target sequence involved can be identified by using a multiplex assay. This can be facilitated by including an additional, different detection tag in each of the OCPs of the group. In this way, TS-DNA generated from each different OCP, representing each different target sequence, can be individually detected. It is convenient that such multiple assays need be performed only when an initial positive result is found.

[0359] 7. In Situ Detection Using RCA

[0360] In situ detection of target sequences is a powerful application of the disclosed method. For example, open circle probes can be ligated on targets immobilized on a substrate, and incubated in situ with fluorescent precursors during rolling circle replication. The circle will remain topologically trapped on the chromosome unless the DNA is nicked (Nilsson et al. (1994)). The resulting TS-DNA will then be associated with the location of the target sequence.

[0361] A useful method of in situ detection uses reporter binding agents having target sequences as the oligonucleotide portion. In this form of the method, reporter binding agents having target sequences as the oligonucleotide portion are associated with target molecules (such as proteins) that are immobilized or otherwise attached to a substrate. Once the reporter binding agent is associated with a target molecule, an open circle probe is hybridized to the target sequence of the reporter binding agent and circularized. The circularized open circle probe is then amplified. The resulting TS-DNA is associated with the site of the target molecule via the open circle probe and reporter binding agent.

[0362] Localization of the TS-DNA for in situ detection can also be enhanced by collapsing the TS-DNA using collapsing detection probes, biotin-antibody conjugates, or both, as described elsewhere herein. Multiplexed in situ detection can be carried out as follows: Rolling circle replication can be carried out using different detection rolling circle replication primers and the same common rolling circle replication primer and secondary DNA strand displacement primer. Detection of different detection rolling circle replication primers (such as by real-time detection using fluorescent change primers) identifies the different targets. Alternatively, rolling circle replication is carried out using unlabeled nucleotides. The different TS-DNAs are then detected using standard multi-color FISH with detection probes specific for each unique target sequence or each unique detection tag in the TS-DNA. Alternatively, and preferably, combinatorial multicolor coding, as described above, can be used for multiplex in situ detection.

[0363] Another method of in situ detection uses reporter binding agents having rolling circle replication primers as the oligonucleotide portion (this is referred to as Reporter Binding Agent Unimolecular Rolling Amplification (RBAURA) in U.S. Pat. No. 6,143,495). In RBAURA, a reporter binding agent is used where the oligonucleotide portion serves as a rolling circle replication primer. Once the reporter binding agent is associated with a target molecule, an amplification target circle is hybridized to the rolling circle replication primer sequence of the reporter binding agent followed by amplification of the ATC by RCA. The resulting TS-DNA has the rolling circle replication primer sequence of the reporter binding agent at one end, thus anchoring the TS-DNA to the site of the target molecule. The rolling circle replication primer sequence can be configured as a fluorescent change primer. Common rolling circle replication primers and secondary DNA strand displacement primers can be used in this form of the method as well. Peptide Nucleic Acid Probe Unimolecular Rolling Amplification (PNAPURA) and Locked Antibody Unimolecular Rolling Amplification (LAURA), described in U.S. Pat. No. 6,143,495, are useful forms of RBAURA and can be adapted to the disclosed method.

[0364] 8. Enzyme-Linked Detection

[0365] Amplified nucleic acid labeled by incorporation of labeled nucleotides can be detected with established enzyme-linked detection systems. For example, amplified nucleic acid labeled by incorporation of biotin-16-UTP (Boehringher Mannheim) can be detected as follows. The nucleic acid is immobilized on a solid glass surface by hybridization with a complementary DNA oligonucleotide (address probe) complementary to the target sequence (or its complement) present in the amplified nucleic acid. After hybridization, the glass slide is washed and contacted with alkaline phosphatase-streptavidin conjugate (Tropix, Inc., Bedford, Mass.). This enzyme-streptavidin conjugate binds to the biotin moieties on the amplified nucleic acid. The slide is again washed to remove excess enzyme conjugate and the chemiluminescent substrate CSPD (Tropix, Inc.) is added and covered with a glass cover slip. The slide can then be imaged in a Biorad Fluorimager.

[0366] 9. Collapse of Nucleic Acids

[0367] Tandem sequence DNA or TS-RNA, which are produced as extended nucleic acid molecules, can be collapsed into a compact structure. It is preferred that the nucleic acid to be collapsed is immobilized on a substrate. A useful means of collapsing nucleic acids is by hybridizing one or more collapsing probes with the nucleic acid to be collapsed. Collapsing probes are oligonucleotides having a plurality of portions each complementary to sequences in the nucleic acid to be collapsed. These portions are referred to as complementary portions of the collapsing probe, where each complementary portion is complementary to a sequence in the nucleic acid to be collapsed. The sequences in the nucleic acid to be collapsed are referred to as collapsing target sequences. The complementary portion of a collapsing probe can be any length that supports specific and stable hybridization between the collapsing probe and the collapsing target sequence. For this purpose, a length of 10 to 35 nucleotides is preferred, with a complementary portion of a collapsing probe 16 to 20 nucleotides long being most preferred. It is preferred that at least two of the complementary portions of a collapsing probe be complementary to collapsing target sequences which are separated on the nucleic acid to be collapsed or to collapsing target sequences present in separate nucleic acid molecules. This allows each detection probe to hybridize to at least two separate collapsing target sequences in the nucleic acid sample. In this way, the collapsing probe forms a bridge between different parts of the nucleic acid to be collapsed. The combined action of numerous collapsing probes hybridizing to the nucleic acid will be to form a collapsed network of cross-linked nucleic acid. Collapsed nucleic acid occupies a much smaller volume than free, extended nucleic acid, and includes whatever detection probe or detection label hybridized to the nucleic acid. This result is a compact and discrete nucleic acid structure which can be more easily detected than extended nucleic acid. Collapsing nucleic acids is useful both for in situ hybridization applications and for multiplex detection because it allows detectable signals to be spatially separate even when closely packed. Collapsing nucleic acids is especially useful for use with combinatorial multicolor coding. Collapsing probes can also contain any of the detection labels described above. Collapsing probes can also be fluorescent change probes. TS-DNA collapse can also be accomplished through the use of ligand/ligand binding pairs (such as biotin and avidin) or hapten/antibody pairs. Nucleic acid collapse is further described in U.S. Pat. No. 6,143,495.

[0368] H. Reporter Binding Agents with Target Sequences

[0369] A useful form of the disclosed method uses reporter binding agents having target sequences as the oligonucleotide portion. The oligonucleotide portion of the reporter binding agent serves as a target sequence. The affinity portion of the reporter binding agent is a specific binding molecule specific for a target molecule of interest, such as proteins or peptides. The reporter binding agent is associated with the target molecule and detection of this interaction is mediated by rolling circle amplification. Unbound reporter binding agents can be removed by washing. Once the reporter binding agent is associated with a target molecule, a open circle probe is hybridized to the target sequence of the reporter binding agent, ligated, and amplified. The resulting TS-DNA is associated with the ligated open circle probe, thus associating the TS-DNA to the site of the target molecule.

[0370] Reporter binding agents are preferably used with a solid-state substrate and in combination with combinatorial multicolor coding. For this purpose, samples to be tested are incorporated into a solid-state sample, as described above. The solid-state substrate is preferably a glass slide and the solid-state sample preferably incorporates up to 256 individual target or assay samples arranged in dots. Multiple solid-state samples can be used to either test more individual samples, or to increase the number of distinct target sequences to be detected. In the later case, each solid-state sample has an identical set of samples dots, and the assay will be carried out using a different set of reporter binding agents and open circle probes, collectively referred to as a probe set, for each solid-state sample. This allows a large number of individuals and target sequences to be assayed in a single assay. By using up to six different labels, combinatorial multicolor coding allows up to 63 distinct targets to be detected on a single solid-state sample. When using multiple solid-state substrates and performing RCA with a different set of reporter binding agents and open circle probes for each solid-state substrate, the same labels can be used with each solid-state sample (although differences between OCPs in each set may require the use of different detection probes). For example, 10 replica slides, each with 256 target sample dots, can be subjected to RCA using 10 different sets of reporter binding agents and open circle probes, where each set is designed for combinatorial multicolor coding of 63 targets. This results in an assay for detection of 630 different target molecules.

[0371] After rolling circle amplification, a cocktail of detection probes is added, where the cocktail contains color combinations that are specific for each OCP. The design and combination of such detection probes for use in combinatorial multicolor coding is described elsewhere herein. It is preferred that the OCPs be designed with combinatorially coded detection tags to allow use of a single set of singly labeled detection probes. It is also preferred that collapsing detection probes be used.

[0372] I. Transcription Following RCA

[0373] Once TS-DNA is generated using RCA, further amplification can be accomplished by transcribing the TS-DNA from promoters embedded in the TS-DNA. This combined process, referred to as rolling circle replication with transcription (RCT), or ligation mediated rolling circle replication with transcription (LM-RCT), requires that the OCP or ATC from which the TS-DNA is made have a promoter portion in its spacer region. The promoter portion is then amplified along with the rest of the OCP or ATC resulting in a promoter embedded in each tandem repeat of the TS-DNA. Because transcription, like rolling circle amplification, is a process that can go on continuously (with re-initiation), multiple transcripts can be produced from each of the multiple promoters present in the TS-DNA. RCT effectively adds another level of amplification of ligated OCP sequences.

[0374] Generally, RCT can be accomplished by performing RCA to produce TS-DNA, and then mixing RNA polymerase with the reaction mixture and incubating under conditions promoting transcription of the tandem sequence DNA. The OCP or ATC must include the sequence of a promoter for the RNA polymerase (a promoter portion) in its spacer region for RCT to work. The transcription step in RCT generally can be performed using established conditions for in vitro transcription of the particular RNA polymerase used. Alternatively, transcription can be carried out simultaneously with rolling circle replication. This is accomplished by mixing RNA polymerase with the reaction mixture prior to rolling circle replication. Transcription can follow any DNA replication operation, such as RCA, LM-RCA, nested LM-RCA, DNA strand displacement, or strand displacement cascade amplification.

[0375] The transcripts generated in RCT can be labeled and/or detected using the same labels, labeling methods, and detection methods described for use with TS-DNA. Most of these labels and methods are adaptable for use with nucleic acids in general. A preferred method of labeling RCT transcripts is by direct labeling of the transcripts by incorporation of labeled nucleotides, most preferably biotinylated nucleotides, during transcription.

[0376] J. Gap-Filling Ligation

[0377] The gap space formed by an OCP hybridized to a target sequence is normally occupied by one or more gap oligonucleotides as described above. Such a gap space may also be filled in by a gap-filling DNA polymerase during the ligation operation. As an alternative, the gap space can be partially bridged by one or more gap oligonucleotides, with the remainder of the gap filled using DNA polymerase. This modified ligation operation is referred to herein as gap-filling ligation and is a preferred form of the ligation operation. The principles and procedure for gap-filling ligation are generally analogous to the filling and ligation performed in gap LCR (Wiedmann et al., PCR Methods and Applications (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY, 1994) pages S51-S64; Abravaya et al., Nucleic Acids Res., 23(4):675-682 (1995); European Patent Application EP0439182 (1991)). In the case of LM-RCA, the gap-filling ligation operation is substituted for the normal ligation operation. Gap-filling ligation provides a means for discriminating between closely related target sequences. Gap-filling ligation can be accomplished by using a different DNA polymerase, referred to herein as a gap-filling DNA polymerase. Suitable gap-filling DNA polymerases are described above. Alternatively, DNA polymerases in general can be used to fill the gap when a stop base is used. The use of stop bases in the gap-filling operation of LCR is described in European Patent Application EP0439182. The principles of the design of gaps and the ends of flanking probes to be joined, as described in EP0439182, is generally applicable to the design of the gap spaces and the ends of target probe portions described herein. Gap-filling ligation is further described in U.S. Pat. No. 6,143,495.

[0378] K. Reporter Binding Agent Unimolecular Rolling Amplification

[0379] Reporter Binding Agent Unimolecular Rolling Amplification (RBAURA) is a form of RCA where a reporter binding agent provides the rolling circle replication primer for amplification of an amplification target circle. In RBAURA, the oligonucleotide portion of the reporter binding agent serves as a rolling circle replication primer. The rolling circle replication primer can be a hairpin rolling circle replication primer. RBAURA allows RCA to produce an amplified signal (that is, TS-DNA) based on association of the reporter binding agent to a target molecule. The specific primer sequence that is a part of the reporter binding agent provides the link between the specific interaction of the reporter binding agent to a target molecule (via the affinity portion of the reporter binding agent) and RCA. In RBAURA, once the reporter binding agent is associated with a target molecule, an amplification target circle is hybridized to the rolling circle replication primer sequence of the reporter binding agent, followed by amplification of the ATC by RCA. The resulting TS-DNA incorporates the rolling circle replication primer sequence of the reporter binding agent at one end, thus anchoring the TS-DNA to the site of the target molecule. RBAURA is a preferred RCA method for in situ detections. For this purpose, it is preferred that the TS-DNA is collapsed using collapsing detection probes, biotin-antibody conjugates, or both, as described above. RBAURA can be performed using any target molecule. Preferred target molecules are nucleic acids, including amplified nucleic acids such as TS-DNA and amplification target circles, antigens and ligands. Examples of the use of such target molecules are described in U.S. Pat. No. 6,143,495. Peptide Nucleic Acid Probe Unimolecular Rolling Amplification (PNAPURA) and Locked Antibody Unimolecular Rolling Amplification (LAURA), described in U.S. Pat. No. 6,143,495, are preferred forms of RBAURA.

[0380] L. Discrimination Between Closely Related Target Sequences

[0381] Open circle probes, gap oligonucleotides, and gap spaces can be designed to discriminate closely related target sequences, such as genetic alleles. Where closely related target sequences differ at a single nucleotide, it is preferred that open circle probes be designed with the complement of this nucleotide occurring at one end of the open circle probe, or at one of the ends of the gap oligonucleotide(s). Where gap-filling ligation is used, it is preferred that the distinguishing nucleotide appear opposite the gap space. This allows incorporation of alternative (that is, allelic) sequence into the ligated OCP without the need for alternative gap oligonucleotides. Where gap-filling ligation is used with a gap oligonucleotide(s) that partially fills the gap, it is preferred that the distinguishing nucleotide appear opposite the portion of gap space not filled by a gap oligonucleotide. Ligation of gap oligonucleotides with a mismatch at either terminus is extremely unlikely because of the combined effects of hybrid instability and enzyme discrimination. When the TS-DNA is generated, it will carry a copy of the gap oligonucleotide sequence that led to a correct ligation. Gap oligonucleotides may give even greater discrimination between related target sequences in certain circumstances, such as those involving wobble base pairing of alleles. Features of open circle probes and gap oligonucleotides that increase the target-dependency of the ligation operation are generally analogous to such features developed for use with the ligation chain reaction. These features can be incorporated into open circle probes and gap oligonucleotides for use in LM-RCA. In particular, European Patent Application EP0439182 describes several features for enhancing target-dependency in LCR that can be adapted for use in LM-RCA. The use of stop bases in the gap space, as described in European Patent Application EP0439182, is a preferred mode of enhancing the target discrimination of a gap-filling ligation operation.

[0382] A preferred form of target sequence discrimination can be accomplished by employing two types of open circle probes. In one embodiment, a single gap oligonucleotide is used which is the same for both target sequences, that is, the gap oligonucleotide is complementary to both target sequences. In a preferred embodiment, a gap-filling ligation operation can be used (Example 3 in U.S. Pat. No. 6,143,495). Target sequence discrimination would occur by virtue of mutually exclusive ligation events, or extension-ligation events, for which only one of the two open-circle probes is competent. Preferably, the discriminator nucleotide would be located at the penultimate nucleotide from the 3′ end of each of the open circle probes. The two open circle probes would also contain two different detection tags designed to bind alternative detection probes and/or address probes. Each of the two detection probes would have a different detection label. Both open circle probes would have the same primer complement portion. Thus, both ligated open circle probes can be amplified using a single primer. Upon array hybridization, each detection probe would produce a unique signal, for example, two alternative fluorescence colors, corresponding to the alternative target sequences.

[0383] These technique for target sequence discrimination are especially useful within matched open circle probe sets.

[0384] M. Size Classes of Tandem Sequence DNA

[0385] Rolling circle amplification can be engineered to produce TS-DNA of different lengths in an assay involving multiple ligated OCPs or ATCs. This can be useful for extending the number of different targets that can be detected in a single assay. TS-DNA of different lengths can be produced in several ways. In one embodiment, the base composition of the spacer region of different classes of OCP or ATC can be designed to be rich in a particular nucleotide. Then a small amount of the dideoxy nucleotide complementary to the enriched nucleotide can be included in the rolling circle amplification reaction. After some amplification, the dideoxy nucleotides will terminate extension of the TS-DNA product of the class of OCP or ATC enriched for the complementary nucleotide. Other OCPs or ATCs will be less likely to be terminated, since they are not enriched for the complementary nucleotide, and will produce longer TS-DNA products, on average.

[0386] In another embodiment, two different classes of OCP or ATC can be designed with different primer complement portions. These different primer complement portions are designed to be complementary to a different rolling circle replication primer. Then the two different rolling circle replication primers are used together in a single rolling circle amplification reaction, but at significantly different concentrations. The primer at high concentration immediately primes rolling circle replication due to favorable kinetics, while the primer at lower concentration is delayed in priming due to unfavorable kinetics. Thus, the TS-DNA product of the class of OCP or ATC designed for the primer at high concentration will be longer than the TS-DNA product of the class of OCP or ATC designed for the primer at lower concentration since it will have been replicated for a longer period of time. These and other techniques for producing size classes of TS-DNA are described in U.S. Pat. No. 6,143,495.

Specific Embodiment

[0387] Disclosed is a method of amplifying nucleic acid sequences, the method comprising a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, and wherein the ligation operation is carried out in the presence of a set of open circle probes. The amplification operation comprises rolling circle replication of the circularized open circle probes, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. The set of open circle probes comprises a plurality of different open circle probes, wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, and wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. Each detection rolling circle replication primer is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes, and wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0388] Also disclosed is a method of amplifying nucleic acid sequences, the method comprising a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, wherein the ligation operation is carried out in the presence of a set of open circle probes. The amplification operation comprises rolling circle replication of the circularized open circle probes, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. The set of open circle probes comprises a plurality of different open circle probes. Each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

[0389] Also disclosed is a method of amplifying nucleic acid sequences, the method comprising an amplification operation, wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles, wherein the amplification operation comprises rolling circle replication of the amplification target circles, wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer. Each detection rolling circle replication primer corresponds to a different amplification target circle in the set of amplification target circles, wherein the secondary DNA strand displacement primer corresponds to all of the amplification target circles in the set of amplification target circles, wherein the common rolling circle replication primer corresponds to all of the amplification target circles in the set of amplification target circles.

[0390] Also disclosed is a method of amplifying nucleic acid sequences, the method comprising a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes. The amplification operation comprises rolling circle replication of the circularized open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

[0391] Also disclosed is a method of amplifying nucleic acid sequences, the method comprising a DNA ligation operation and an amplification operation, wherein the DNA ligation operation comprises circularization of one or more open circle probes, wherein the amplification operation comprises rolling circle replication of the circularized open circle probes, wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers, and wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.

[0392] Also disclosed is a method of amplifying nucleic acid sequences, the method comprising an amplification operation, wherein the amplification operation comprises rolling circle replication of the amplification target circles, wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers, wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.

[0393] Also disclosed is a method of selectively amplifying nucleic acid sequences related to one or more target sequences, the method comprising,

[0394] (a) mixing a set of open circle probes with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture,

[0395] (b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles,

[0396] (c) mixing a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture, and

[0397] (d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles.

[0398] The set of open circle probes comprises a plurality of different open circle probes, wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set,

[0399] wherein the amplification target circles formed from the open circle probes in the set of open circle probes comprise a set of amplification target circles. Each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes. Replication of the amplification target circles results in the formation of tandem sequence DNA.

[0400] In the disclosed method, each detection rolling circle replication primer can comprise a complementary portion, wherein each open circle probe can comprise a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer can be complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond. The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0401] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The amplification operation can result in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0402] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0403] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

[0404] Each peptide nucleic acid fluor can comprise a different fluorescent moiety. The amplification operation can result in disassociation of the peptide nucleic acid flours from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce. The detection rolling circle replication primer can be a hairpin quenched primer.

[0405] The ligation operation can be carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes can comprise a plurality of different open circle probes. Each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer can correspond to all of the open circle probes in all of the sets of open circle probes, wherein the common rolling circle replication primer can correspond to all of the open circle probes in all of the sets of open circle probes. Each detection rolling circle replication primer can comprise a complementary portion, a fluorescent moiety, and a quencher complement portion. Each detection rolling circle replication primer corresponding to an open circle probe in the same set of open circle probes can comprise a different fluorescent moiety.

[0406] At least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes can comprise the same fluorescent moiety as at least one of the detection rolling circle replication primers in a different one of the sets of open circle probes. At least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes can comprise the same fluorescent moiety as a different detection rolling circle replication primer in the same set of open circle probes. Each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes, wherein the common rolling circle replication primer can correspond to all of the open circle probes in all of the sets of open circle probes. The amplification operation can be carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer can correspond to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer can correspond to all of the open circle probes in a given set of open circle probes.

[0407] Each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer can correspond to all of the open circle probes in all of the sets of open circle probes. The amplification operation can be carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer can correspond to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer can correspond to all of the open circle probes in a given set of open circle probes. Each detection rolling circle replication primer can correspond to a different open circle probe in all of the sets of open circle probes. The amplification operation can be carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer can correspond to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer can correspond to all of the open circle probes in a given set of open circle probes. The amplification operation can be carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer can correspond to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer can correspond to all of the open circle probes in a given set of open circle probes.

[0408] All of the open circle probes in all of the sets of open circle probes can be different. Each detection rolling circle replication primer can correspond to a different open circle probe in a given set of open circle probes. At least one of the detection rolling circle replication primers can correspond to an open circle probe in each of at least two of the sets of open circle probes. At least one of the detection rolling circle replication primers can correspond to an open circle probe in each of at least two of the sets of open circle probes.

[0409] The peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety. Each detection rolling circle replication primer can comprise a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primers via the quencher complement portion. Each detection rolling circle replication primer can comprise a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the amplification operation can result in disassociation of the peptide nucleic acid quencher from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce. The peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety. Each detection rolling circle replication primer can comprise a complementary portion, a quenching moiety, and a quencher complement portion, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primers via the quencher complement portion.

[0410] The open circle probes in the matched open circle probe set can be targeted to different forms of the same target sequence. The different forms of the same target sequence can comprise a wild type form of the target sequence and a mutant form of the target sequence. The different forms of the same target sequence can further comprise a second mutant form of the target sequence. The different forms of the same target sequence can further comprise a plurality of different mutant forms of the target sequence. The different forms of the same target sequence can comprise a plurality of different mutant forms of the target sequence. The different forms of the same target sequence can comprise a normal form of the target sequence and a mutant form of the target sequence. The different forms of the same target sequence can further comprise a second mutant form of the target sequence. The different forms of the same target sequence can further comprise a plurality of different mutant forms of the target sequence.

[0411] The set of open circle probes can comprise a plurality of matched open circle probe sets. The open circle probes in each of the matched open circle probe sets can be targeted to different forms of the same target sequence, and open circle probes in different matched open circle probe sets can be targeted to different target sequences. The different forms of the same target sequence can comprise a wild type form of the target sequence and a mutant form of the target sequence. The different forms of the same target sequence can further comprise a second mutant form of the target sequence. The different forms of the same target sequence can further comprise a plurality of different mutant forms of the target sequence. The different forms of the same target sequence can comprise a plurality of different mutant forms of the target sequence. The different forms of the same target sequence can comprise a normal form of the target sequence and a mutant form of the target sequence. The different forms of the same target sequence can further comprise a second mutant form of the target sequence. The different forms of the same target sequence can further comprise a plurality of different mutant forms of the target sequence. The different target sequences can be in the same gene. The different target sequences can be associated with the same disease or condition.

[0412] The matched open circle probe set can consist of two open circle probes, wherein one of the open circle probes in the matched open circle probe set can be targeted to a wild type form of the target sequence, wherein the other open circle probe in the matched open circle probe set can be targeted to a mutant form of the target sequence. Each detection rolling circle replication primer can comprise a different fluorescent moiety. Each detection rolling circle replication primer corresponding to an open circle probes in the matched open circle probe set can comprise a different fluorescent moiety.

[0413] The method can further comprise, following the ligation operation, heating the circularized open circle probes. The circularized open circle probes can be heated to about 95° C. for about 10 minutes.

[0414] The open circle probes each can be specific for a target sequence, wherein each target sequence can comprise a 5′ region and a 3′ region, wherein each open circle probe can comprise a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule can comprise, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl-group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence. At least one of the target sequences can further comprise a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

[0415] The ligation operation can comprise mixing the open circle probes and one or more gap oligonucleotides with one or more target samples, and incubating under conditions that promote hybridization between the open circle probes and the gap oligonucleotides and the target sequences, and ligation of the open circle probes and gap oligonucleotides to form the circularized open circle probes. Each gap oligonucleotide can comprise a single-stranded, linear DNA molecule comprising a 5′ phosphate group and a 3′ hydroxyl group, wherein each gap oligonucleotide can be complementary all or a portion of the central region of the target sequence. A complement to the central region of the target sequence can be synthesized during the ligation operation. A plurality of the open circle probes each can be specific for a different target sequence. A plurality of different target sequences can be detected. The amplification operation can produce amplified nucleic acid, wherein the method can further comprise detecting the amplified nucleic acid with one or more detection probes. A portion of each of a plurality of the detection probes can have sequence matching or complementary to a portion of a different one of the open circle probes, wherein a plurality of different amplified nucleic acids can be detected using the plurality of detection probes.

[0416] The spacer portion can comprise a detection primer complement portion. The spacer portion can comprise a common primer complement portion. The intramolecular stem structure of at least one of the open circle probes can form a stem and loop structure. A portion of one of the target probe portions of at least one of the open circle probes can be in the loop of the stem and loop structure, wherein the portion of the target probe portion in the loop can hybridize to the target sequence, wherein hybridization of the target probe portion in the loop to the target sequence disrupts the intramolecular stem structure. A hybrid between the target sequence and the target probe portion at the end of the open circle probes that can form an intramolecular stem structure can be more stable than the intramolecular stem structure.

[0417] In the method, if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure can be extended during the amplification operation using the open circle probe as a template. The intramolecular stem structure can form under the conditions used for the amplification operation. The intramolecular stem structure can prevent the open circle probes from priming nucleic acid replication. The intramolecular stem structure can prevent the open circle probes from serving as a template for rolling circle replication. The intramolecular stem structure can form a hairpin structure. The intramolecular stem structure can form a stem and loop structure. One of the ends of the open circle probes can be a 3′ end, wherein the 3′ end of at least one of the open circle probes can form an intramolecular stem structure.

[0418] Rolling circle replication can be primed by one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer can comprise two ends, wherein at least one of the ends of at least one of the detection rolling circle replication primers can form an intramolecular stem structure, wherein priming by the detection rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the detection rolling circle replication primers to the circularized open circle probes. The amplification operation can produce tandem sequence DNA, wherein the amplification operation can further comprise secondary DNA strand displacement. Rolling circle replication can be primed by one or more common rolling circle replication primers, wherein each common rolling circle replication primer can comprise two ends, wherein at least one of the ends of at least one of the common rolling circle replication primers can form an intramolecular stem structure, wherein priming by the common rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the common rolling circle replication primers to the circularized open circle probes.

[0419] The amplification operation can produce tandem sequence DNA, wherein the method can further comprises detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0420] Each detection rolling circle replication primer can comprise a complementary portion, wherein each open circle probe can comprise a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer can be complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

[0421] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0422] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each detection rolling circle replication primer can comprise a different fluorescent moiety.

[0423] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor can comprise a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0424] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each peptide nucleic acid fluor can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a hairpin quenched primer.

[0425] The ligation operation can be carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes can comprise a plurality of different open circle probes. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer can comprise a fluorescent moiety. Each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer can comprise a quenching moiety.

[0426] Two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set can be targeted to different forms of the same target sequence. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The method can further comprise, following the ligation operation, heating the circularized open circle probes.

[0427] Each open circle probe can comprises two ends, wherein the open circle probes each can be specific for a target sequence, wherein each target sequence can comprise a 5′ region and a 3′ region, wherein each open circle probe can comprise a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule can comprise, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group. The left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence. At least one of the target sequences can further comprise a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

[0428] Each open circle probe can comprises two end, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure. Each open circle probe can comprise two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. If one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure can be extended during the amplification operation using the open circle probe as a template.

[0429] The amplification operation can produce tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0430] Each detection rolling circle replication primer can comprise a complementary portion, wherein each amplification target circle can comprise a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer can be complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.

[0431] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0432] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same.

[0433] The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor can comprise a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0434] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles. Each peptide nucleic acid fluor can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a hairpin quenched primer.

[0435] The amplification operation can be carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles can comprise a plurality of different amplification target circles. Each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer can comprise a fluorescent moiety. Each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer can comprise a quenching moiety.

[0436] The amplification operation can produce tandem sequence DNA, wherein the method can further comprises detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0437] The amplification operation can be carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer can comprise a complementary portion, wherein each open circle probe can comprise a detection primer complement portion, wherein each detection rolling circle replication primer can correspond to a different open circle probe in the set of open circle probes, wherein the complementary portion of the detection rolling circle replication primer can be complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

[0438] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0439] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same.

[0440] The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor can comprise a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0441] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same.

[0442] The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each peptide nucleic acid fluor can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a hairpin quenched primer. The ligation operation can be carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes can comprise a plurality of different open circle probes. The amplification operation can be carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer can comprise a fluorescent moiety.

[0443] The amplification operation can be carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer can comprise a quenching moiety. The open circle probes in the matched open circle probe set can be targeted to different forms of the same target sequence. The amplification operation can be carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer can comprise a different fluorescent moiety.

[0444] The method can further comprise, following the ligation operation, heating the circularized open circle probes. Each open circle probe can comprise two ends, wherein the open circle probes each can be specific for a target sequence, wherein each target sequence can comprise a 5′ region and a 3′ region, wherein each open circle probe can comprise a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule can comprise, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence. At least one of the target sequences can further comprise a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

[0445] Each open circle probe can comprise two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure. Each open circle probe can comprise two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. If one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure can be extended during the amplification operation using the open circle probe as a template.

[0446] The amplification operation can produce tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0447] Each detection rolling circle replication primer can comprise a complementary portion, wherein each open circle probe can comprise a detection primer complement portion, wherein the ligation operation can be carried out in the presence of a set of open circle probes, wherein the set of open circle probes can comprise a plurality of different open circle probes, wherein the complementary portion of the detection rolling circle replication primer can be complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

[0448] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0449] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same.

[0450] The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor can comprise a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0451] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same.

[0452] The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes. Each peptide nucleic acid fluor can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a hairpin quenched primer.

[0453] The ligation operation can be carried out in the presence of a plurality of sets of open circle probes, wherein each set of open circle probes can comprise a plurality of different open circle probes. The peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer can comprise a fluorescent moiety. The peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

[0454] The ligation operation can be carried out in the presence of a set of open circle probes, wherein the set of open circle probes can comprise a plurality of different open circle probes, wherein two or more of the open circle probes in the set of open circle probes can constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence. Each detection rolling circle replication primer can comprise a different fluorescent moiety. The method can further comprise, following the ligation operation, heating the circularized open circle probes.

[0455] Each open circle probe can comprise two ends, wherein the open circle probes each can be specific for a target sequence, wherein each target sequence can comprise a 5′ region and a 3′ region, wherein each open circle probe can comprise a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule can comprise, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence. At least one of the target sequences can further comprise a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

[0456] Each open circle probe can comprise two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure. Each open circle probe can comprise two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence. If one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure can be extended during the amplification operation using the open circle probe as a template.

[0457] The amplification operation can produce tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0458] Each detection rolling circle replication primer can comprise a complementary portion, wherein each amplification target circle can comprise a detection primer complement portion, wherein the amplification operation can be carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles can comprise a plurality of different amplification target circles, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.

[0459] The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher can comprise a quenching moiety. Association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

[0460] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles can be the same, and the peptide nucleic acid quencher associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.

[0461] Each detection rolling circle replication primer can comprise a different fluorescent moiety. The detection rolling circle replication primer can be a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer can further comprise a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer can be associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor can be associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor can comprise a fluorescent moiety. Association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

[0462] The quencher complement portion of each detection rolling circle replication primer can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers. The quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles can be the same, and the peptide nucleic acid fluor associated with each detection rolling circle replication primer can be the same. The quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles can be different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles. Each peptide nucleic acid fluor can comprise a different fluorescent moiety.

[0463] The detection rolling circle replication primer can be a hairpin quenched primer. The amplification operation can be carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles can comprise a plurality of different amplification target circles. The amplification operation can be carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles can comprise a plurality of different amplification target circles. The peptide nucleic acid quencher can comprise peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer can comprise a fluorescent moiety.

[0464] The peptide nucleic acid fluor can comprise peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer can comprise a quenching moiety. The amplification operation can produce tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA. The tandem sequence DNA can be detected via one or more fluorescent change probes. The fluorescent change probes can be hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination. The tandem sequence DNA can be detected via one or more fluorescent change primers. The fluorescent change primers can be stem quenched primers, hairpin quenched primers, or a combination. The amplification operation can produce tandem sequence DNA and secondary tandem sequence DNA, wherein the method can further comprise detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

[0465] Also disclosed is a kit for selectively detecting one or more target sequences or selectively amplifying nucleic acid sequences related to one or more target sequences, the kit comprising, a set of open circle probes each comprising two ends, wherein at least one of the ends of one of the open circle probe can form an intramolecular stem structure, wherein portions of each open circle probe are complementary to the one or more target sequences; a plurality of detection rolling circle replication primers, wherein all or a portion of each detection rolling circle replication primer is complementary to a portion of one or more of the open circle probes; one or more secondary DNA strand displacement primers, wherein all or a portion of each secondary DNA strand displacement primer matches a portion of one or more of the open circle probes; and one or more common rolling circle replication primers, wherein all or a portion of each common rolling circle replication primer is complementary to a portion of one or more of the open circle probes.

[0466] All or a portion of each detection rolling circle replication primer is complementary to a portion of a different one or more of the open circle probes in the set of open circle probes, all or a portion of each secondary DNA strand displacement primer matches a portion of all of the open circle probes in the set of open circle probes, and all or a portion of each common rolling circle replication primer is complementary to a portion of all of the open circle probes in the set of open circle probes. The end of the open circle probe that can form an intramolecular stem structure can be a 3′ end. Each target sequence can comprise a 5′ region and a 3′ region, wherein the open circle probes each can comprise a single-stranded, linear DNA molecule comprising, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the spacer portion can comprise a primer complement portion, wherein the left target probe portion can be complementary to the 3′ region of at least one of the target sequences and the right target probe portion can be complementary to the 5′ region of the same target sequence, wherein the rolling circle replication primer can comprise a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes.

[0467] At least one target sequence can further comprise a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion nor the right target probe portion of the open circle probe complementary to the target sequence is complementary to the central region of the target sequence. The kit can further comprise one or more gap oligonucleotides, wherein the gap oligonucleotides can be complementary to all or a portion of the central region of the target sequence. The target probe portions of the open circle probes can be complementary to a different target sequence for each of a plurality of the open circle probes.

[0468] The kit can further comprise one or more reporter binding agents each comprising a specific binding molecule and an oligonucleotide portion, wherein the oligonucleotide portion can comprise one of the target sequences. The portions of the open circle probes that are complementary to the target sequence can be complementary to a different target sequence for each of a plurality of the open circle probes.

Examples

A. Example 1

Primer Extension Assay

[0469] This example demonstrates that a hairpin open circle probe with a 5′ overhanging end can be extended from the 3′ end. Such extension would lead to an inactive open circle probe. Open circle probe 1822ocT was used as a model for the new design of 3′ hairpin open circle probes. The new design was compared to the conventional design.

[0470] Conventional OCP design sequence: 5′-GAAGAACTGGACAGATTT ACTACGTATGTTGACTGGTCACACGTCGTTCTAGTACGCTTCTACTCCCTCTT GAGATGTTCTGCTTTGTT 3′ (SEQ ID NO:9)

[0471] New (hairpin) OCP design sequence: 5′-GAAGAACTGGA CAGATTTACTACGTATGTTGACTGGTCACACGTCGTTCTAGTAACAAAGCAC TCCCTCTTGAGATGTTCTGCTTTGTT 3′ (SEQ ID NO:10)

[0472] &ggr;32P ATP exchange reaction end labeling: M13 bacteriophage forward primer and OCPs were end labeled with radioactive 32P in an exchange reaction as follows:

[0473] 30 minutes exchange reactions were carried out in 20 ill volume containing 10 &mgr;l of M13 bacteriophage forward primer or open circle probe DNA (1 pM/&mgr;l), 4 &mgr;l 5×exchange buffer (Gibco BRL, cat # 10456-010) (250 mM imidazole-HCl (pH 6.4), 60 mM MgCl2, 5 mM 2-mercaptoethanol, and 0.35 mM ADP), 0.5 &mgr;l T4 polynucleotide kinase (NEB) (10u/&mgr;l), 2.5 &mgr;l dH2O, and 3 &mgr;l 10 mM &ggr;32P ATP (10 &mgr;Ci/&mgr;i). DNA was purified and eluted into 100 &mgr;l of EB buffer (10 mM Tris-HCl, pH 8.5) using QIAquick nucleotide removal kit (QIAGEN).

[0474] Annealing of &ggr;32P ATP labeled forward primer to M13 DNA: Annealing reaction was carried out by mixing: 3.3 &mgr;l forward primer, 7.6 &mgr;M13 mp19 ssDNA (0.25 &mgr;g/&mgr;l), 1 &mgr;l Tris-HCl (pH 7.54), and 18.1 &mgr;l dH2O. Heated to 95° C. for 2 minutes, and cooled to room temperature.

[0475] M13 ladder preparation: 4 &mgr;l of the above reaction was added to 1 &mgr;l 0.1 M DTT, 7.6 &mgr;l 50×sequenase buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl2), 7.6 &mgr;l dH2O, and 0.4 &mgr;l sequenase (USB) (4 units/&mgr;l). The mix was divided into 4 equal portions and 2.5 &mgr;l of ddG, ddA, ddT, or ddC was added into each portion. All four portions were then incubated at 37° C. for 5 minutes.

[0476] Primer extension reactions: 30 &mgr;l extension reactions were performed as follows: 0.1 &mgr;l of labeled open circle probe was added to ligation and ERCA reaction mix containing: 1 &mgr;l Ampligase buffer (20 mM Tris-HCl, pH 8.3) (Epicentre Technologies), 3 &mgr;l 10×modified ThermoPol reaction buffer (200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4 and 1%Triton X-100), 3 &mgr;l 50% TMA oxalate, 20.7 &mgr;l dH2O, 1.2 &mgr;l 10 mM dNTP mix (dATP, dCTP, dGTP, and TTP), and 1 &mgr;l Bst polymerase (8 units/&mgr;l) (New England Biolabs, Mass.), the mix was incubated at 60° C.

[0477] 3 &mgr;l Aliquots were pipetted out between 15 seconds and 2 hr time points and added into 3 &mgr;l 2×stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). Samples were then boiled for 5 minutes and electrophoresed on a 8% denaturing polyacrylamide gel. For each open circle probe, aliquot number one was taken prior to adding the polymerase enzyme, which represents the unextended open circle probe.

[0478] Results: A hairpin forms at the 3′ end of the open circle probe that allowed the ERCA DNA polymerase to extend 54 bases from the self-annealed 3′ end of the open circle probe. Full extension should have converted the hairpin open circle probe to an inert double-stranded form. This reaction, called the “suicide pathway”, inactivates the open circle probe. The reaction was completed within 15 sec of the start of primer extension reaction.

B. Example 2

VCAM Rolling Circle Amplification Assay

[0479] This example describes single nucleotide polymorphism (SNP) detection on genomic DNA, using exponential rolling circle amplification (ERCA). Specifically, Exponential Rolling Circle Amplification is used for allele discrimination on genomic DNA on an ABI Prism 7700 Sequence Detection System using generic P1 Amplifluors as detection probes.

[0480] Oligonucleotide sequences: 4 VCAMinA sequence: 5′-AAATTGATTCAGGAAATACTAGCTTATAAAGACTCGTCATGTCTCAGCTCTAGTTTCTGATCCCATGACTTCAC (SEQ ID NO:11) CTACCAAATATCTAGGGATCAGAA-3′ VCAMocG sequence: 5′-AAATTGATTCAGGAAATACTAGCTTATAAAATGTTGACT GGT CAC ACG TCGCTCTGATCCC ATG ACT (SEQ ID NO:12) TCA CCT ACC AAA TAT CTA GGG ATC AGA G-3′ VCAMinA P2: CTTCACCTACCAAATATCTAGGGATCAGAA (SEQ ID NO:13) VCAMocG P21: CTTCACCTACCAAATATCTAGGGATCAGAG (SEQ ID NO:14) P1 in Amplifluor: 5′-FAM-TCGATGACTGACGGTCATCG-Dabcyl-dT)-ACTAGAGCTGAGACATGACGAGTC-3′ (SEQ ID NO:15) P1 oc Amplifluor: 5′-TET-TCGATGACTGACGGTCATCG-(Dabcyl-dT)-ACGACGTGTGACCAGTCAACAT-3′ (SEQ ID NO:16)

[0481] Primer (P1) is an Amplifluor (a type of fluorescent change primer) and is complementary to the region of the spacer region of an open circle probe. The sequence of the allele-specific primer (P2) is homologous to the 3′ arm of an open circle probe. The Amplifluor P1s have either FAM or TET fluorophores at the 5′ ends for the two alleles. The Tms of both these primers is approximately 65° C.

[0482] DNA Annealing and Ligation: The reactions were set up in 96-well MicroAmp Optical plates (Perkin Elmer) in a 10 &mgr;l reaction volume containing 1 unit Ampligase (Epicentre Technologies), 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, and 0.01% Triton®X-100. Standard reactions contained 0.01 nM open circle probes and 100 ng of Alu I digested genomic DNA. DNA was denatured by heating the reactions at 95° C. for 3 min followed by annealing and ligation at 60° C. for 30 min.

[0483] ERCA™ Reaction: For each 30 &mgr;l reaction to be run, 20 &mgr;l of ERCA mix was added to the 10 &mgr;l ligation mix. ERCA mix was prepared as follows: 3 &mgr;l of 10×Bst Thermopol buffer (200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4 and 1% Triton X-100) containing no Mg2+, 3 &mgr;l 50 mM TMA oxalate, 1.2 &mgr;l 10 mM dNTP mix (dATP, dGTP, dCTP, and dTTP), 3 &mgr;l 10 &mgr;M Amplifluor P1 primer, 3 &mgr;l 10 &mgr;M P2 primer, 2.5 &mgr;l 20 &mgr;M ROX dye, 1 &mgr;l of 8 units/&mgr;l Bst polymerase (New England Biolabs, MA), and 3.3 &mgr;l water. 20 &mgr;l of ERCA mix was added to the 10 &mgr;l ligation reaction. Real time ERCA reaction was performed in 96-well MicroAmp Optical Plates (Perkin-Elmer) and run on real time ABI Prism 7700 (Perkin-Elmer) for 3 hrs. Specific signal was expressed as “delta Ct”. Delta Ct=(Ct minus ligase control—Ct plus ligase).

[0484] Results: Genotyping assays with the hairpin open circle probe for the SNP VCAM gave a typical real time profiles, and 98% overall accuracy (Table 1) when 92 genomic DNA samples were analyzed. 5 TABLE 1 Genotype Accuracy % Accuracy CT 31/31 100.0% TT (FAM-A) 55/56  98.2% CC (TET-G) 4/5  80.0% Total 90/92   98%

C. Example 3

Hemochromatosis H63D Mutation Genotyping Using Rolling Circle Amplification

[0485] This example describes an example of the disclosed method using a matched set of open circle probes, detection rolling circle primers that are fluorescent change primers (specifically, peptide nucleic acid quenched primers) via association with a peptide nucleic acid quencher, a common rolling circle replication primer, and a secondary DNA strand displacement primer.

[0486] 1. Oligonucleotides

[0487] The oligonucleotides used are described below. The oligonucleotides have a 5′ phosphate unless a different molecule or moiety is indicated.

[0488] An open circle probe targeted to a wild type sequence (OCP H63D 1166/1704-2 wt#10 3′mm short): 6 (SEQ ID NO:17) 5′-ATC ATA GAA CAC GAA CAG CTG GTC ATC CAG TTC TTC GCT GCC CAT CGC GCA GAC ACG ATA CAA GAG AGT GAC TCT CTT G

[0489] An open circle probe targeted to a mutant form of the same sequence (OCP H63D 5901/1704-2 mut#5): 7 (SEQ ID NO:18) 5′-ATC ATA GAA CAC GAA CAG CTG GTC ATC TGC TCT GTT ATC GGC CGT CGC GCA GAC ACG ATA GAT GAG GCG ACT CTC ATC

[0490] A peptide nucleic acid quencher (Q-PNA-13), which is annealed to the detection rolling circle replication primers:

[0491] Ac-X-OO-tga-ttg-cga-atg-Lys(Dabcyl) (SEQ ID NO: 1)

[0492] A detection rolling circle replication primer (P1 Cy3 5901), which is annealed to the peptide nucleic acid quencher (Q-PNA-13) and which corresponds to the mutant open circle probe (OCP H63D 5901/1704-2 mut#5):

[0493] 5′-/Cy3/Tc att cgc aat ca ACG GCC GAT AAC AGA (SEQ ID NO:3)

[0494] A detection rolling circle replication primer (P1 FAM 1166), which is annealed to the peptide nucleic acid quencher (Q-PNA-13) and which corresponds to the wild type open circle probe (OCP H63D 1166/1704-2 wt#10 3′mm short):

[0495] 5′-/6-FAM/Tcattcgcaatca ATG GGC AGC GAA GAA (SEQ ID NO:19)

[0496] A secondary DNA strand displacement primer (P2 1704) which corresponds to both open circle probes:

[0497] 5′-CGC GCA GACACG ATA-3′ (SEQ ID NO:4)

[0498] A common rolling circle replication primer (3C H63D) which corresponds to both open circle probes:

[0499] 5′-TG TTC GTG TTC TAT GAT-3′ (SEQ ID NO:20)

[0500] The sequence relationships between these oligonucleotides is shown as follows. Complementary sequences between the open circle probes and the detection rolling circle replication primers are shown in bold. Complementary sequences between the common rolling circle replication primer and the open circle probes is underlined. Matching sequences between the secondary DNA strand displacement primer and the open circle probes is shown in italic. Complementary sequence between the detection rolling circle replication primers and the peptide nucleic acid quencher is shown as lowercase.

[0501] These oligonucleotides use many of the features that can be used in the disclosed method. Both open circle probes can form an intramolecular stem structure (in the form of a hairpin). The open circle probes constitute a matched open circle probe set since they are targeted to different forms (wild type and mutant) of the same target sequence (hemochromatosis H63D sequence). Both detection rolling circle amplification primers contain the same 5′ tail that anneals to the same peptide nucleic acid quencher (Q-PNA-13). A common rolling circle replication primer (3C H63D) that is complementary to both open circle probes is an extra non-fluorescent primer used to suppress unwanted background ERCA. A secondary DNA strand displacement primer (P2 1704) has sequence matching sequence in both open circle probes. The reaction is performed with two OCPs in the same reaction.

[0502] 2. OCP Annealing and Ligation

[0503] MicroAmp Optical plates and/or tubes were set up on ice for the required number of reactions. Ligation reaction volume was 10 &mgr;l. 200 ng (50 ng/&mgr;l) of genomic DNA or multiple displacement amplification (MDA) product to be genotyped was added to the appropriate tubes. DNA from 34 homozygous wild type individuals, 25 heterozygous wild type/mutant individuals, and 12 homozygous mutant individuals was used. A premix was prepared for 1.5 times the number of ligation reactions, containing 0.1 unit of Ampligase and 1 &mgr;l of 10×Ampligase reaction buffer (Epicentre) per reaction. OCPs were added to a final concentration of 50 pM each.: Sufficient water was added to bring the total reaction volume to 10 &mgr;l per reaction upon addition of premix. Using eppendorf repeat pipettor, 6 &mgr;l premix was dispensed into wells containing genomic DNA. Samples were incubated as follows: 95° C. for 30 seconds, 63° C. for 20 minutes, 95° C. for 10 minutes, 4° C. hold. The ligation reactions were kept at 4° C. until ERCA mix was added.

[0504] 3. Exponential Rolling Circle Amplification (ERCA) Reaction

[0505] ERCA master mix was set up the in a PCR enclosure to reduce the possibility of cross-contamination. A premix of 1.3 times the number of reactions was prepared, such that the reaction volume added to the ligation reaction was 20 pi. Each ERCA reaction contained 3 &mgr;l of 10×Bst Thermopol II buffer (NEB), 3 &mgr;l 50 mM TMA oxalate, 1.2 &mgr;l 10 mM dNTPs, 1 &mgr;l of 8u/&mgr;l Bst polymerase. Final concentration of P1 FAM 1166 primer was 0.3 &mgr;M; P1 Cy3 5901 primer was 0.4 &mgr;M, P2 1704 primer was 0.525 &mgr;M, Q-PNA-13 was 1.4 &mgr;M, and 3C H63D primer was 0.3 &mgr;M. The volume was brought to 20 &mgr;l per ERCA reaction using water. An eppendorf repeat pipettor was used to dispense 20 &mgr;l of the ERCA master mix into the wells containing the 10 &mgr;l ligation reaction. The reactions were incubated at 60° C. in BioRad I-Cycler, collecting data using both the FAM and Cy3 channels.

[0506] All of the wild type individuals gave a strong FAM signal (corresponding to the wild type open circle probe) and essentially no Cy3 signal (corresponding to the mutant open circle probe). This is the expected result from wild type individuals (who have two copies of the wild type sequence). Similarly, all of the mutant individuals gave essentially no FAM signal (corresponding to the wild type open circle probe) and a strong Cy3 signal (corresponding to the mutant open circle probe). This is the expected result from mutant individuals (who have two copies of the mutant sequence). Finally, all of the heterozygous individuals gave a moderate FAM signal (corresponding to the wild type open circle probe) and a moderate Cy3 signal (corresponding to the mutant open circle probe). This is the expected result from heterozygous individuals (who have both the mutant and wild type sequences).

D. Example 4

Factor II Prothrombin Mutation Genotyping Using Rolling Circle Amplification

[0507] This example describes an example of the disclosed method using a matched set of open circle probes, detection rolling circle primers that are fluorescent change primers (specifically, peptide nucleic acid quenched primers) via association with a peptide nucleic acid quencher, a common rolling circle replication primer, and a secondary DNA strand displacement primer.

[0508] 1. Oligonucleotides

[0509] The oligonucleotides used are described below. The oligonucleotides have a 5′ phosphate unless a different molecule or moiety is indicated.

[0510] An open circle probe targeted to a wild type sequence (FII Wild type): 8 (SEQ ID NO:21) 5′-AGCCTCAATGCTCCCAGTGCACAAGACCGAAAGGGTAGTCGCGG ATTGTTGCGCTGAGAAATAAAAGTGACTCTCAGCG

[0511] An open circle probe targeted to a mutant form of the same sequence (FII Mutant): 9 (SEQ ID NO:22) 5′-AGCCTCAATGCTCCCAGTGCACTCAATCCCAGGCGAGTCGCGG ATTGTTGTGCTGAGAGAATAAAAGTGACTCTCAGCA

[0512] A peptide nucleic acid quencher (Q-PNA-13), which is annealed to the detection rolling circle replication primers:

[0513] Ac-X-OO-tgattgcgaatg-Lys(Dabcyl) (SEQ ID NO: 1)

[0514] A detection rolling circle replication primer (FII Mutant P1), which is annealed to the peptide nucleic acid quencher (Q-PNA-13) and which corresponds to the mutant open circle probe (FII Mutant):

[0515] 5′-/Cy3/tcattcgcaatcaCGCCTGGGATTGAGT (SEQ ID NO:23)

[0516] A detection rolling circle replication primer (FII Wild type P1), which is annealed to the peptide nucleic acid quencher (Q-PNA-13) and which corresponds to the wild type open circle probe (FII Wild type):

[0517] 5′-/6-FAM/tcattcgcaatcaCCCTTTCGGTCTTGT (SEQ ID NO:24)

[0518] A secondary DNA strand displacement primer (FII P2) which corresponds to both open circle probes:

[0519] 5′-AGTCGCGGATTGTTG-3′ (SEQ ID NO:25)

[0520] A common rolling circle replication primer (FII allele specific primer) which corresponds to both open circle probes:

[0521] 5′-CACTGGGAGCATTGA-3′ (SEQ ID NO:26)

[0522] The sequence relationships between these oligonucleotides is shown as follows. Complementary sequences between the open circle probes and the detection rolling circle replication primers are shown in bold. Complementary sequences between the common rolling circle replication primer and the open circle probes is underlined. Matching sequences between the secondary DNA strand displacement primer and the open circle probes is shown in italic. Complementary sequence between the detection rolling circle replication primers and the peptide nucleic acid quencher is shown as lowercase.

[0523] These oligonucleotides use many of the features that can be used in the disclosed method. Both open circle probes can form an intramolecular stem structure (in the form of a hairpin). The open circle probes constitute a matched open circle probe set since they are targeted to different forms (wild type and mutant) of the same target sequence (Factor II prothrombin sequence). Both detection rolling circle amplification primers contain the same 5′ tail that anneals to the same peptide nucleic acid quencher (Q-PNA-13). A common rolling circle replication primer (FII allele specific primer) that is complementary to both open circle probes is an extra non-fluorescent primer used to suppress unwanted background ERCA. A secondary DNA strand displacement primer (FII P2) has sequence matching sequence in both open circle probes. The reaction is performed with two OCPs in the same reaction.

[0524] 2. OCP Annealing and Ligation

[0525] 50 ng to 1 &mgr;g of either genomic DNA or of an MDA amplified genomic sample was mixed with both OCPs (typically 0.5 nM final concentration for each OCP) and 0.5 unit of Ampligase (Epicentre Technologies, Madison, Wis.) in 1×Ampligase buffer (Epicentre), for a total volume of 10 &mgr;l. The reaction was heated to 95° C. for 10 seconds, and cooled to 63-68° C. for 5-20 minutes, during which time the OCP annealed to genomic target and was circularized by ligase.

[0526] 3. Exponential Rolling Circle Amplification (ERCA) Reaction

[0527] The ligation reaction was heated to 95° C. for 10 minutes to release ligated circles from genomic DNA. The reaction was cooled to 4° C., and 20 &mgr;l ERCA reaction mix was added (typically 16 units BST polymerase (New England Biolabs, Beverly, Mass.), 6 mM dNTPs, 0.5 &mgr;M FII Wild type P1, 0.7 &mgr;M FII Mutant P1, 0.5 &mgr;M FII P2, 4 &mgr;M FII allele specific primer, 4 &mgr;M Q-PNA-13, 7.5 &mgr;M TMA oxalate in 1×ThermoPol Buffer II, all concentrations final). Reactions were incubated at 60° C. for 3 hours in an I-Cycler (BioRad, Hercules, Calif.) reading both FAM and Cy3 channels. Signals typically appeared after 10-20 minutes.

[0528] 4. Results and Analysis

[0529] The basic results (fluorescence signal over time) are shown in FIG. 6. FIG. 6A shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single normal human sample. FIG. 6B shows Cy3 fluorescence from the same 32 samples in FIG. 6A. FIG. 6C shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single heterozygous human sample. FIG. 6D shows Cy3 fluorescence from the same 32 samples in FIG. 6C. FIG. 6E shows FAM fluorescence in amplification reactions of nucleic acid samples from 32 repeats of a single homozygous mutant human sample. FIG. 6F shows Cy3 fluorescence from the same 32 samples in FIG. 6E.

[0530] As can be seen, all of the wild type individuals gave a strong FAM signal (corresponding to the wild type open circle probe; FIG. 6A) and essentially no Cy3 signal (corresponding to the mutant open circle probe; FIG. 6D). This is the expected result from wild type individuals (who have two copies of the wild type sequence). Similarly, all of the mutant individuals gave essentially no FAM signal (corresponding to the wild type open circle probe; FIG. 6C) and a strong Cy3 signal (corresponding to the mutant open circle probe; FIG. 6F). This is the expected result from mutant individuals (who have two copies of the mutant sequence). Finally, all of the heterozygous individuals gave a moderate FAM signal (corresponding to the wild type open circle probe; FIG. 6B) and a moderate Cy3 signal (corresponding to the mutant open circle probe; FIG. 6E). This is the expected result from heterozygous individuals (who have both the mutant and wild type sequences).

[0531] The genotype for each sample was determined by amplitude of amplification. The average amplification threshold time for all amplified reactions was determined using the I-Cycler software. Fluorescence traces were normalized using early cycles as a baseline, and a threshold value was determined, typically at 10-fold above the average standard deviation of the baseline values. Threshold cycle for each trace was measured at the point where the trace crossed the threshold value. Threshold cycle values fell into three distinct clusters, one each for homozygous normal, heterozygous, and homozygous mutant.

[0532] Genotype of each sample was determined automatically using a modified fuzzy c-means clustering algorithm (Pickering, J., et al., Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms. Nucleic Acids Res, 30(12):e60 (2002)), which groups the data into three genotypes plus a negative control, and assigns a confidence level to each genotyping call from 0 (not in cluster) to 1 (100% certainty that point belongs to cluster). The results of this clustering can be seen in FIG. 5. FIG. 5 is an X-Y plot of end point fluorescent readings obtained from the samples in FIGS. 6A-6F. The X-axis shows Cy3 fluorescence (arbitrary units) corresponding to the mutant genotype. The Y-axis shows FAM signal corresponding to wild type genotype, also in arbitrary units. End point readings fall into three clusters that are easily differentiated by genotype, as indicated in the Figure.

[0533] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0534] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a rolling circle replication primer” includes a plurality of such rolling circle replication primers, reference to “the open circle probes” is a reference to one or more open circle probes and equivalents thereof known to those skilled in the art, and so forth. “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0535] Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0536] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0537] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of amplifying nucleic acid sequences, the method comprising

a DNA ligation operation and an amplification operation,

wherein the DNA ligation operation comprises circularization of one or more open circle probes,

wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes,

wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence,

wherein the amplification operation comprises rolling circle replication of the circularized open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer,

wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor,

wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes,

wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

2. The method of claim 1 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

3. The method of claim 2 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

4. The method of claim 3 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

5. The method of claim 3 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

6. The method of claim 3 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

7. The method of claim 3 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

8. The method of claim 3 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

9. The method of claim 3 wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

10. The method of claim 2 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

11. The method of claim 10 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

12. The method of claim 10 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

13. The method of claim 10 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

14. The method of claim 10 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

15. The method of claim 14 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

16. The method of claim 10 wherein the amplification operation results in disassociation of the peptide nucleic acid flours from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

17. The method of claim 2 wherein the detection rolling circle replication primer is a hairpin quenched primer.

18. The method of claim 1 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.

19. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in all of the sets of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in all of the sets of open circle probes.

20. The method of claim 19 each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion.

21. The method of claim 20 wherein each detection rolling circle replication primer corresponding to an open circle probe in the same set of open circle probes comprises a different fluorescent moiety.

22. The method of claim 21 wherein at least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes comprises the same fluorescent moiety as at least one of the detection rolling circle replication primers in a different one of the sets of open circle probes.

23. The method of claim 20 wherein at least one of the detection rolling circle replication primers corresponding to an open circle probe in one of the sets of open circle probes comprises the same fluorescent moiety as a different detection rolling circle replication primer in the same set of open circle probes.

24. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in all of the sets of open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer corresponds to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer corresponds to all of the open circle probes in a given set of open circle probes.

25. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in all of the sets of open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer corresponds to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer corresponds to all of the open circle probes in a given set of open circle probes.

26. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in all of the sets of open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of secondary DNA strand displacement primers, wherein each secondary DNA strand displacement primer corresponds to open circle probes in a different set of open circle probes, wherein a single secondary DNA strand displacement primer corresponds to all of the open circle probes in a given set of open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of common rolling circle replication primers, wherein each common rolling circle replication primer corresponds to open circle probes in a different set of open circle probes, wherein a single common rolling circle replication primer corresponds to all of the open circle probes in a given set of open circle probes.

27. The method of claim 18 wherein all of the open circle probes in all of the sets of open circle probes are different.

28. The method of claim 18 wherein each detection rolling circle replication primer corresponds to a different open circle probe in a given set of open circle probes.

29. The method of claim 28 wherein at least one of the detection rolling circle replication primers corresponds to an open circle probe in each of at least two of the sets of open circle probes.

30. The method of claim 18 wherein at least one of the detection rolling circle replication primers corresponds to an open circle probe in each of at least two of the sets of open circle probes.

31. The method of claim 1 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety.

32. The method of claim 31 wherein each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primers via the quencher complement portion.

33. The method of claim 31 wherein each detection rolling circle replication primer comprises a complementary portion, a fluorescent moiety, and a quencher complement portion, wherein the amplification operation results in disassociation of the peptide nucleic acid quencher from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

34. The method of claim 1 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety.

35. The method of claim 34 wherein each detection rolling circle replication primer comprises a complementary portion, a quenching moiety, and a quencher complement portion, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primers via the quencher complement portion.

36. The method of claim 1 wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.

37. The method of claim 36 wherein the different forms of the same target sequence comprise a wild type form of the target sequence and a mutant form of the target sequence.

38. The method of claim 37 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.

39. The method of claim 37 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.

40. The method of claim 36 wherein the different forms of the same target sequence comprise a plurality of different mutant forms of the target sequence.

41. The method of claim 36 wherein the different forms of the same target sequence comprise a normal form of the target sequence and a mutant form of the target sequence.

42. The method of claim 41 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.

43. The method of claim 41 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.

44. The method of claim 36 wherein the set of open circle probes comprises a plurality of matched open circle probe sets.

45. The method of claim 44 wherein the open circle probes in each of the matched open circle probe sets are targeted to different forms of the same target sequence, wherein open circle probes in different matched open circle probe sets are targeted to different target sequences.

46. The method of claim 45 wherein the different forms of the same target sequence comprise a wild type form of the target sequence and a mutant form of the target sequence.

47. The method of claim 46 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.

48. The method of claim 46 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.

49. The method of claim 45 wherein the different forms of the same target sequence comprise a plurality of different mutant forms of the target sequence.

50. The method of claim 45 wherein the different forms of the same target sequence comprise a normal form of the target sequence and a mutant form of the target sequence.

51. The method of claim 50 wherein the different forms of the same target sequence further comprise a second mutant form of the target sequence.

52. The method of claim 50 wherein the different forms of the same target sequence further comprise a plurality of different mutant forms of the target sequence.

53. The method of claim 45 wherein the different target sequences are in the same gene.

54. The method of claim 45 wherein the different target sequences are associated with the same disease or condition.

55. The method of claim 36 wherein the matched open circle probe set consists of two open circle probes, wherein one of the open circle probes in the matched open circle probe set is targeted to a wild type form of the target sequence, wherein the other open circle probe in the matched open circle probe set is targeted to a mutant form of the target sequence.

56. The method of claim 36 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

57. The method of claim 36 wherein each detection rolling circle replication primer corresponding to an open circle probes in the matched open circle probe set comprises a different fluorescent moiety.

58. The method of claim 1 further comprising, following the ligation operation, heating the circularized open circle probes.

59. The method of claim 58 wherein the circularized open circle probes are heated to about 95° C. for about 10 minutes.

60. The method of claim 1 wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′ region and a 3′ region, wherein each open circle probe comprises a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule comprises, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence.

61. The method of claim 60 wherein at least one of the target sequences further comprises a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

62. The method of claim 61 wherein the ligation operation comprises mixing the open circle probes and one or more gap oligonucleotides with one or more target samples, and incubating under conditions that promote hybridization between the open circle probes and the gap oligonucleotides and the target sequences, and ligation of the open circle probes and gap oligonucleotides to form the circularized open circle probes,

wherein each gap oligonucleotide comprises a single-stranded, linear DNA molecule comprising a 5′ phosphate group and a 3′ hydroxyl group, wherein each gap oligonucleotide is complementary all or a portion of the central region of the target sequence.

63. The method of claim 61 wherein a complement to the central region of the target sequence is synthesized during the ligation operation.

64. The method of claim 60 wherein a plurality of the open circle probes are each specific for a different target sequence.

65. The method of claim 64 wherein a plurality of different target sequences are detected.

66. The method of claim 64 wherein the amplification operation produces amplified nucleic acid, wherein the method further comprises

detecting the amplified nucleic acid with one or more detection probes.

67. The method of claim 66 wherein a portion of each of a plurality of the detection probes has sequence matching or complementary to a portion of a different one of the open circle probes, wherein a plurality of different amplified nucleic acids are detected using the plurality of detection probes.

68. The method of claim 60 wherein the spacer portion comprises a detection primer complement portion.

69. The method of claim 60 wherein the spacer portion comprises a common primer complement portion.

70. The method of claim 60 wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.

71. The method of claim 60 wherein a portion of one of the target probe portions of at least one of the open circle probes is in the loop of the stem and loop structure, wherein the portion of the target probe portion in the loop can hybridize to the target sequence, wherein hybridization of the target probe portion in the loop to the target sequence disrupts the intramolecular stem structure.

72. The method of claim 60 wherein a hybrid between the target sequence and the target probe portion at the end of the open circle probes that can form an intramolecular stem structure is more stable than the intramolecular stem structure.

73. The method of claim 1 wherein if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.

74. The method of claim 1 wherein the intramolecular stem structure can form under the conditions used for the amplification operation.

75. The method of claim 1 wherein the intramolecular stem structure prevents the open circle probes from priming nucleic acid replication.

76. The method of claim 1 wherein the intramolecular stem structure prevents the open circle probes from serving as a template for rolling circle replication.

77. The method of claim 1 wherein the intramolecular stem structure forms a hairpin structure.

78. The method of claim 1 wherein the intramolecular stem structure forms a stem and loop structure.

79. The method of claim 1 wherein one of the ends of the open circle probes is a 3′ end, wherein the 3′ end of at least one of the open circle probes can form an intramolecular stem structure.

80. The method of claim 1 wherein rolling circle replication is primed by one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer comprises two ends, wherein at least one of the ends of at least one of the detection rolling circle replication primers can form an intramolecular stem structure, wherein priming by the detection rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the detection rolling circle replication primers to the circularized open circle probes.

81. The method of claim 1 wherein the amplification operation produces tandem sequence DNA, wherein the amplification operation further comprises secondary DNA strand displacement.

82. The method of claim 1 wherein rolling circle replication is primed by one or more common rolling circle replication primers, wherein each common rolling circle replication primer comprises two ends, wherein at least one of the ends of at least one of the common rolling circle replication primers can form an intramolecular stem structure, wherein priming by the common rolling circle replication primers that can form an intramolecular stem structure is dependent on hybridization of the common rolling circle replication primers to the circularized open circle probes.

83. The method of claim 1 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

84. The method of claim 83 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

85. The method of claim 84 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

86. The method of claim 83 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

87. The method of claim 86 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

88. The method of claim 1 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

89. A method of amplifying nucleic acid sequences, the method comprising

a DNA ligation operation and an amplification operation,

wherein the DNA ligation operation comprises circularization of one or more open circle probes,

wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes,

wherein the amplification operation comprises rolling circle replication of the circularized open circle probes,

wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer,

wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes.

90. The method of claim 89 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

91. The method of claim 90 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

92. The method of claim 91 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

93. The method of claim 91 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

94. The method of claim 91 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

95. The method of claim 91 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

96. The method of claim 91 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

97. The method of claim 90 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

98. The method of claim 97 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

99. The method of claim 97 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

100. The method of claim 97 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

101. The method of claim 97 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

102. The method of claim 101 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

103. The method of claim 90 wherein the detection rolling circle replication primer is a hairpin quenched primer.

104. The method of claim 89 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.

105. The method of claim 89 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.

106. The method of claim 89 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

107. The method of claim 89 wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.

108. The method of claim 107 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

109. The method of claim 89 further comprising, following the ligation operation, heating the circularized open circle probes.

110. The method of claim 89 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′ region and a 3′ region, wherein each open circle probe comprises a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule comprises, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence, wherein at least one of the target sequences further comprises a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

111. The method of claim 89 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.

112. The method of claim 89 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence,

wherein if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.

113. The method of claim 89 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

114. The method of claim 113 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

115. The method of claim 114 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

116. The method of claim 113 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

117. The method of claim 116 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

118. The method of claim 89 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

119. A method of amplifying nucleic acid sequences, the method comprising

an amplification operation,

wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles,

wherein the amplification operation comprises rolling circle replication of the amplification target circles,

wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer,

wherein each detection rolling circle replication primer corresponds to a different amplification target circle in the set of amplification target circles, wherein the secondary DNA strand displacement primer corresponds to all of the amplification target circles in the set of amplification target circles, wherein the common rolling circle replication primer corresponds to all of the amplification target circles in the set of amplification target circles.

120. The method of claim 119 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each amplification target circle comprises a detection primer complement portion, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.

121. The method of claim 120 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

122. The method of claim 121 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

123. The method of claim 121 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

124. The method of claim 121 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

125. The method of claim 121 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.

126. The method of claim 121 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

127. The method of claim 120 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

128. The method of claim 127 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

129. The method of claim 127 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

130. The method of claim 127 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

131. The method of claim 127 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.

132. The method of claim 131 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

133. The method of claim 120 wherein the detection rolling circle replication primer is a hairpin quenched primer.

134. The method of claim 119 wherein the amplification operation is carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles comprises a plurality of different amplification target circles.

135. The method of claim 119 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.

136. The method of claim 119 wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

137. The method of claim 119 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

138. The method of claim 137 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

139. The method of claim 138 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

140. The method of claim 137 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

141. The method of claim 140 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

142. The method of claim 119 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

143. A method of amplifying nucleic acid sequences, the method comprising

a DNA ligation operation and an amplification operation,

wherein the DNA ligation operation comprises circularization of one or more open circle probes,

wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes,

wherein the amplification operation comprises rolling circle replication of the circularized open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set.

144. The method of claim 143 wherein the amplification operation is carried out in the presence of a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer, wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

145. The method of claim 144 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

146. The method of claim 145 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

147. The method of claim 145 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

148. The method of claim 145 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

149. The method of claim 145 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

150. The method of claim 145 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

151. The method of claim 144 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

152. The method of claim 151 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

153. The method of claim 151 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

154. The method of claim 151 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

155. The method of claim 151 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

156. The method of claim 155 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

157. The method of claim 144 wherein the detection rolling circle replication primer is a hairpin quenched primer.

158. The method of claim 143 wherein the ligation operation is carried out in the presence of one or more additional sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.

159. The method of claim 143 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.

160. The method of claim 143 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

161. The method of claim 143 wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.

162. The method of claim 161 wherein the amplification operation is carried out in the presence of one or more detection rolling circle replication primers, wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

163. The method of claim 143 further comprising, following the ligation operation, heating the circularized open circle probes.

164. The method of claim 143 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′ region and a 3′ region, wherein each open circle probe comprises a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule comprises, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence, wherein at least one of the target sequences further comprises a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

165. The method of claim 143 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.

166. The method of claim 143 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence,

wherein if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.

167. The method of claim 143 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

168. The method of claim 167 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

169. The method of claim 168 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

170. The method of claim 167 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

171. The method of claim 170 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

172. The method of claim 143 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

173. A method of amplifying nucleic acid sequences, the method comprising

a DNA ligation operation and an amplification operation,

wherein the DNA ligation operation comprises circularization of one or more open circle probes,

wherein the amplification operation comprises rolling circle replication of the circularized open circle probes,

wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers,

wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.

174. The method of claim 173 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each open circle probe comprises a detection primer complement portion, wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the open circle probe to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an open circle probe to which the detection rolling circle replication primer does not correspond.

175. The method of claim 174 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

176. The method of claim 175 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

177. The method of claim 175 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

178. The method of claim 175 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

179. The method of claim 175 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

180. The method of claim 175 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

181. The method of claim 174 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

182. The method of claim 181 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

183. The method of claim 181 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

184. The method of claim 181 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an open circle probe in the set of open circle probes is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

185. The method of claim 181 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an open circle probe in the set of open circle probes.

186. The method of claim 185 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

187. The method of claim 174 wherein the detection rolling circle replication primer is a hairpin quenched primer.

188. The method of claim 173 wherein the ligation operation is carried out in the presence of a plurality of sets of open circle probes, wherein each set of open circle probes comprises a plurality of different open circle probes.

189. The method of claim 173 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.

190. The method of claim 173 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

191. The method of claim 173 wherein the ligation operation is carried out in the presence of a set of open circle probes, wherein the set of open circle probes comprises a plurality of different open circle probes, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set, wherein the open circle probes in the matched open circle probe set are targeted to different forms of the same target sequence.

192. The method of claim 191 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

193. The method of claim 173 further comprising, following the ligation operation, heating the circularized open circle probes.

194. The method of claim 173 wherein each open circle probe comprises two ends, wherein the open circle probes are each specific for a target sequence, wherein each target sequence comprises a 5′ region and a 3′ region, wherein each open circle probe comprises a single-stranded, linear DNA molecule, wherein the single-stranded, linear DNA molecule comprises, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the left target probe portion is complementary to the 3′ region of the target sequence, wherein the right target probe portion is complementary to the 5′ region of the target sequence, wherein at least one of the target sequences further comprises a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion of the open circle probe specific for the target sequence nor the right target probe portion of the open circle probe specific for the target sequence is complementary to the central region of the target sequence.

195. The method of claim 173 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein the intramolecular stem structure of at least one of the open circle probes forms a stem and loop structure.

196. The method of claim 173 wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence,

wherein if one or more of the open circle probes that can form an intramolecular stem structure are not circularized, the end of at least one of the uncircularized open circle probes that forms the intramolecular stem structure is extended during the amplification operation using the open circle probe as a template.

197. The method of claim 173 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

198. The method of claim 197 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

199. The method of claim 198 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

200. The method of claim 197 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

201. The method of claim 200 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

202. The method of claim 173 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

203. A method of amplifying nucleic acid sequences, the method comprising

an amplification operation,

wherein the amplification operation comprises rolling circle replication of the amplification target circles,

wherein the amplification operation is carried out in the presence of one or more rolling circle replication primers,

wherein at least one of the rolling circle replication primers is associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor.

204. The method of claim 203 wherein each detection rolling circle replication primer comprises a complementary portion, wherein each amplification target circle comprises a detection primer complement portion, wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles, wherein the complementary portion of the detection rolling circle replication primer is complementary to the detection primer complement portion of the amplification target circle to which the detection rolling circle replication primer corresponds, wherein the complementary portion of the detection rolling circle replication primer is not substantially complementary to an amplification target circle to which the detection rolling circle replication primer does not correspond.

205. The method of claim 204 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a fluorescent moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein the peptide nucleic acid quencher is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid quencher comprises a quenching moiety, wherein association of the peptide nucleic acid quencher with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid quenchers from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the detection rolling circle replication primers to fluoresce.

206. The method of claim 205 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

207. The method of claim 205 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

208. The method of claim 205 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid quencher associated with each detection rolling circle replication primer is the same.

209. The method of claim 205 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.

210. The method of claim 205 wherein each detection rolling circle replication primer comprises a different fluorescent moiety.

211. The method of claim 204 wherein the detection rolling circle replication primer is a peptide nucleic acid quenched primer, wherein the detection rolling circle replication primer further comprises a quenching moiety and a quencher complement portion, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid fluor, wherein the peptide nucleic acid fluor is associated with the detection rolling circle replication primer via the quencher complement portion, wherein the peptide nucleic acid fluor comprises a fluorescent moiety, wherein association of the peptide nucleic acid fluor with the detection rolling circle replication primer quenches fluorescence from the fluorescent moiety, wherein the amplification operation results in disassociation of the peptide nucleic acid fluors from the detection rolling circle replication primers, thereby allowing the fluorescent moiety of the peptide nucleic acid fluors to fluoresce.

212. The method of claim 211 wherein the quencher complement portion of each detection rolling circle replication primer is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

213. The method of claim 211 wherein the quencher complement portion of at least one of the detection rolling circle replication primers is different from the quencher complement portion of at least one of the other detection rolling circle replication primers.

214. The method of claim 211 wherein the quencher complement portion of each detection rolling circle replication primer corresponding to an amplification target circle in the set of amplification target circles is the same, wherein the peptide nucleic acid fluor associated with each detection rolling circle replication primer is the same.

215. The method of claim 211 wherein the quencher complement portion of at least one of the detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles is different from the quencher complement portion of at least one of the other detection rolling circle replication primers corresponding to an amplification target circle in the set of amplification target circles.

216. The method of claim 215 wherein each peptide nucleic acid fluor comprises a different fluorescent moiety.

217. The method of claim 204 wherein the detection rolling circle replication primer is a hairpin quenched primer.

218. The method of claim 203 wherein the amplification operation is carried out in the presence of a set of amplification target circles, wherein the set of amplification target circles comprises a plurality of different amplification target circles.

219. The method of claim 218 wherein the amplification operation is carried out in the presence of one or more additional sets of amplification target circles, wherein each set of amplification target circles comprises a plurality of different amplification target circles.

220. The method of claim 203 wherein the peptide nucleic acid quencher comprises peptide nucleic acid and a quenching moiety, wherein the detection rolling circle replication primer comprises a fluorescent moiety.

221. The method of claim 203 wherein the peptide nucleic acid fluor comprises peptide nucleic acid and a fluorescent moiety, wherein the detection rolling circle replication primer comprises a quenching moiety.

222. The method of claim 203 wherein the amplification operation produces tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA.

223. The method of claim 222 wherein the tandem sequence DNA is detected via one or more fluorescent change probes.

224. The method of claim 223 wherein the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

225. The method of claim 222 wherein the tandem sequence DNA is detected via one or more fluorescent change primers.

226. The method of claim 225 wherein the fluorescent change primers are stem quenched primers, hairpin quenched primers, or a combination.

227. The method of claim 203 wherein the amplification operation produces tandem sequence DNA and secondary tandem sequence DNA, wherein the method further comprises detecting the tandem sequence DNA, the secondary tandem sequence DNA, or both.

228. A method of selectively amplifying nucleic acid sequences related to one or more target sequences, the method comprising,

(a) mixing a set of open circle probes with a target sample, to produce an OCP-target sample mixture, and incubating the OCP-target sample mixture under conditions that promote hybridization between the open circle probes and the target sequences in the OCP-target sample mixture, wherein the set of open circle probes comprises a plurality of different open circle probes,

wherein each open circle probe comprises two ends, wherein at least one of the ends of at least one of the open circle probes can form an intramolecular stem structure, wherein circularization of the open circle probes that can form an intramolecular stem structure is dependent on hybridization of the open circle probe to a target sequence, wherein two or more of the open circle probes in the set of open circle probes constitute a matched open circle probe set,

(b) mixing ligase with the OCP-target sample mixture, to produce a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the open circle probes to form amplification target circles, wherein the amplification target circles formed from the open circle probes in the set of open circle probes comprise a set of amplification target circles,

(c) mixing a plurality of detection rolling circle replication primers, a secondary DNA strand displacement primer, and a common rolling circle replication primer with the ligation mixture, to produce a primer-ATC mixture, and incubating the primer-ATC mixture under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers in the primer-ATC mixture, wherein each detection rolling circle replication primer is associated with a peptide nucleic acid quencher, wherein each detection rolling circle replication primer corresponds to a different open circle probe in the set of open circle probes, wherein the secondary DNA strand displacement primer corresponds to all of the open circle probes in the set of open circle probes, wherein the common rolling circle replication primer corresponds to all of the open circle probes in the set of open circle probes, and

(d) mixing DNA polymerase with the primer-ATC mixture, to produce a polymerase-ATC mixture, and incubating the polymerase-ATC mixture under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA.

229. A kit for selectively detecting one or more target sequences or selectively amplifying nucleic acid sequences related to one or more target sequences, the kit comprising,

a set of open circle probes each comprising two ends, wherein at least one of the ends of one of the open circle probe can form an intramolecular stem structure, wherein portions of each open circle probe are complementary to the one or more target sequences,

a plurality of detection rolling circle replication primers, wherein all or a portion of each detection rolling circle replication primer is complementary to a portion of one or more of the open circle probes,

one or more secondary DNA strand displacement primers, wherein all or a portion of each secondary DNA strand displacement primer matches a portion of one or more of the open circle probes, and

one or more common rolling circle replication primers, wherein all or a portion of each common rolling circle replication primer is complementary to a portion of one or more of the open circle probes.

230. The kit of claim 229 wherein all or a portion of each detection rolling circle replication primer is complementary to a portion of a different one or more of the open circle probes in the set of open circle probes,

wherein all or a portion of each secondary DNA strand displacement primer matches a portion of all of the open circle probes in the set of open circle probes, and

wherein all or a portion of each common rolling circle replication primer is complementary to a portion of all of the open circle probes in the set of open circle probes.

231. The kit of claim 229 wherein the end of the open circle probe that can form an intramolecular stem structure is a 3′ end.

232. The kit of claim 229 wherein each target sequence comprises a 5′ region and a 3′ region, wherein the open circle probes each comprise a single-stranded, linear DNA molecule comprising, from 5′ end to 3′ end, a 5′ phosphate group, a right target probe portion, a spacer portion, a left target probe portion, and a 3′ hydroxyl group, wherein the spacer portion comprises a primer complement portion, wherein the left target probe portion is complementary to the 3′ region of at least one of the target sequences and the right target probe portion is complementary to the 5′ region of the same target sequence, wherein the rolling circle replication primer comprises a single-stranded, linear nucleic acid molecule comprising a complementary portion that is complementary to the primer complement portion of one or more of the open circle probes.

233. The kit of claim 232 wherein at least one target sequence further comprises a central region located between the 5′ region and the 3′ region, wherein neither the left target probe portion nor the right target probe portion of the open circle probe complementary to the target sequence is complementary to the central region of the target sequence.

234. The kit of claim 233 further comprising one or more gap oligonucleotides, wherein the gap oligonucleotides are complementary to all or a portion of the central region of the target sequence.

235. The kit of claim 232 the target probe portions of the open circle probes are complementary to a different target sequence for each of a plurality of the open circle probes.

236. The kit of claim 229 further comprising one or more reporter binding agents each comprising a specific binding molecule and an oligonucleotide portion, wherein the oligonucleotide portion comprises one of the target sequences.

237. The kit of claim 229 wherein the portions of the open circle probes that are complementary to the target sequence are complementary to a different target sequence for each of a plurality of the open circle probes.