Abstract: Disclosed are compositions and methods for sensitive multiplex detection of analytes. The disclosed compositions, referred to as detectors, accomplish this detection by associating specific binding molecules—which interact with desired targets—with block groups in a carrier. The block groups are made up of blocks which, through the combination of different blocks, constitute a code for a given detector. The blocks are detectable and each detector is distinguishable from other detectors by its block group. The coding of the block groups greatly increasing the number of distinguishable detectors from a relatively small number of blocks. The detection burden remains low even with such a large number of block groups because only the blocks need be distinguished from each other during detection. Numerous block molecules of each type making up the block group can be present in the carrier to effectively amplify the signal generated from targets.

Abstract: Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Binary Encoded Sequence Tags (BEST), involves generation of a set of nucleic acid fragments; adding an adaptor to the ends containing recognition site for cleavage at a site offset from the recognition site; cleaving the fragment to generate fragments having a plurality sticky ends; indexing of the fragments into sets based on the sequence of sticky ends. The fragments are indexed by adding a offset adaptor to newly generated ends. A different adaptor will be coupled to each different sticky end. The resulting fragments—which will have defined ends, be of equal lengths (in preferred embodiment), and a central sequence derived from the source nucleic acid molecule—are binary sequence tags. The binary sequence tags can be used and further analyzed in numerous ways.

Abstract: Disclosed are compositions and methods for sensitive detection of one or multiple analytes. In general, the methods involve the use of special label components, referred to as reporter signals, that can be associated with, incorporated into, or otherwise linked to the analytes. In some embodiments, the reporter signals can be altered such that the altered forms of different reporter signals can be distinguished from each other. In some embodiments, sets of reporter signals can be used where two or more of the reporter signals in a set have one or more common properties that allow the reporter signals having the common property to be distinguished and/or separated from other molecules lacking the common property.

Abstract: Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation 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. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences.

Abstract: Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe 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.

Abstract: Disclosed are a composition and a method for a multiplexing-optimized reporter system. The system is designed for the simultaneous detection of dozens or even hundreds of analytes. The analytes may be present on the surface of cells in suspension, on the surface of cytology smears, on the surface of histological sections, on the surface of DNA microarrays, on the surface of protein microarrays, on the surface of beads, or any other situation where complex samples need to be studied. The disclosed composition accomplishes this detection by associating specific binding molecules—which interact with desired targets—with numerous tag molecules in a carrier. The numerous tag molecules can be detected and effectively amplify the signal generated from targets.

Abstract: Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Fixed Address Analysis of Sequence Tags (FAAST), involves generation of a set of nucleic acid fragments having a variety of sticky end sequences; indexing of the fragments into sets based on the sequence of sticky ends; associating a detector sequence with the fragments; sequence-based capture of the indexed fragments on a detector array; and detection of the fragment labels. Generation of the multiple sticky end sequences is accomplished by incubating the nucleic acid sample with one or more nucleic acid cleaving reagents. The indexed fragments are captured by hybridization and coupling, preferably by ligation, to a probe. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner.

Abstract: The present invention provides a replicatable and hybridizable recombinant single-stranded RNA probe molecule comprising: a recognition sequence for the binding of an RNA-directed RNA polymerase; a sequence required for the initiation of product strand synthesis by the polymerase; and a heteroloqus RNA sequence inserted at a specific site in the internal region of the recombinant molecule and complementary to an oligo or polynucleotide of interest. This invention also provides methods for determining the presence of concentration of an oligo- or polynucleotide of interest in a sample and for simultaneously determining the presence or concentration of several different oligo- or polynucleotides of interest in a sample.

Abstract: Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method involves amplifying nucleic acid fragments of interest using a primer that can form a hairpin structure; sequence-based coupling of the amplified fragments to detector probes; and detection of the coupled fragments. The amplified fragments are coupled by hybridization and coupling, preferably by ligation, to detector probes. A hairpin structure formed at the end of the amplified fragments facilitates coupling of the fragments to the probes. The method allows detection of the fragments where detection provides some sequence information for the fragments. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner. The method can also be used to detect amplified fragments having a known sequence.

Abstract: Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Binary Encoded Sequence Tags (BEST), involves generation of a set of nucleic acid fragments; adding an adaptor to the ends containing recognition site for cleavage at a site offset from the recognition site; cleaving the fragment to generate fragments having a plurality sticky ends; indexing of the fragments into sets based on the sequence of sticky ends. The fragments are indexed by adding a offset adaptor to newly generated ends. A different adaptor will be coupled to each different sticky end. The resulting fragments—which will have defined ends, be of equal lengths (in preferred embodiment), and a central sequence derived from the source nucleic acid molecule—are binary sequence tags. The binary sequence tags can be used and further analyzed in numerous ways.

Abstract: Disclosed are reagents and a method for efficient in vitro molecular cloning of nucleic acid molecules of interest. Because the method is entirely in vitro, it can be automated and scaled-up in ways that are not possible in cell-based molecular cloning. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermic reaction that replicates the vector sequences exponentially. The amplification process is amenable to automation where multiple reactions are carried out simultaneously in a small area. The amplified nucleic acid can be used for any purpose and in any manner that nucleic acid cloned or amplified by known methods can be used.

Abstract: Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation 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. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences.

Abstract: Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe 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.

Abstract: Disclosed are compositions and a method for detecting single nucleic acid molecules using rolling circle amplification (RCA) of single-stranded circular templates, referred to as amplification target circles, primed by immobilized primers. In one form of the method, referred to as a bipartite primer rolling circle amplification, (BP-RCA), RCA of the amplification target circle (ATC) depends on the formation of a primer by target-mediated ligation. In the presence of a nucleic acid molecule having the target sequence, a probe and a combination probe/primer oligonucleotide can hybridize to adjacent sites on the target sequence allowing the probes to be ligated together. By attaching the first probe to a substrate such as a bead or glass slide, unligated probe/primer can be removed after ligation. The only primers remaining will be primers ligated, via the probe portion of the probe/primer, to the first probe. The ligated primer can then be used to prime replication of its cognate ATC.

Abstract: Disclosed are reagents and a method for efficient in vitro molecular cloning of nucleic acid molecules of interest. Because the method is entirely in vitro, it can be automated and scaled-up in ways that are not possible in cell-based molecular cloning. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermic reaction that replicates the vector sequences exponentially. The amplification process is amenable to automation where multiple reactions are carried out simultaneously in a small area. The amplified nucleic acid can be used for any purpose and in any manner that nucleic acid cloned or amplified by known methods can be used.

Abstract: Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase.

Abstract: Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Fixed Address Analysis of Sequence Tags (FAAST), involves generation of a set of nucleic acid fragments having a variety of sticky end sequences; indexing of the fragments into sets based on the sequence of sticky ends; associating a detector sequence with the fragments; sequence-based capture of the indexed fragments on a detector array; and detection of the fragment labels. Generation of the multiple sticky end sequences is accomplished by incubating the nucleic acid sample with one or more nucleic acid cleaving reagents. The indexed fragments are captured by hybridization and coupling, preferably by ligation, to a probe. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner.

Abstract: Disclosed are compositions and an in vitro method for cloning and/or amplification of nucleic acid sequences of interest. The method is based on strand displacement replication of the nucleic acid sequences by multiple priming on artificial long terminal repeat (ALTR) sequences appended to the ends of the nucleic acid molecule of interest. The nucleic acid molecules for cloning and amplification can be very long, up to 40 to 80 Kb or longer. In a preferred form of the method, a single primer is used to prime strand displacement replication at multiple sites in artificial long terminal repeat sequences, flanking a target nucleic acid, containing multiple tandem repeats of a primer complement sequence. Amplification proceeds by replication initiated at each primer and continuing through the target nucleic acid sequence. This nested replication of multiple copies significantly increases the amplification yield for extremely long nucleic acid molecules.

Abstract: Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation 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. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences.

Abstract: Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation 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. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences.