Abstract: Compounds are described comprising purines substituted at the C-8 position and pyrimidines substituted at the C-4 position with a linker and quencher. These compounds, when incorporated into hairpin oligonucleotides quench the fluorescence of fluorophores linked to the 5′-terminus of the oligonucleotide. These nucleotide-quencher compounds are also easily incorporated into oligonucleotides using conventional or automated oligonucleotide synthetic techniques.

Abstract: The present invention provides a novel detecting reagent for double-stranded nucleic acid, and a method of using it to detect double-stranded nucleic acid formed by hybridization with a probe, with absolutely no labeling of the target nucleic acid. The detecting reagent for double-stranded nucleic acid of the invention is characterized by comprising, in the same molecule, a naphthalenediimide group which is intercalatable into double-stranded nucleic acid and a &bgr;-diketone group capable of forming a lanthanoid metal complex.

Abstract: Nucleic acid analogs provide a particularly useful tool for the preparation of complex polymeric structures of defined geometry because they are relatively stable to reaction conditions for the preparation of such structures and provide the opportunity to induce reactive groups which would not be possible with usual nucleic acids.

Abstract: The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluorescent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. The invention also provides a method for identifying and isolating rare genes. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.

Abstract: The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.

Abstract: Novel nucleoside or nucleotide analogs comprising 2′-O-amino residues, processes for their synthesis and incorporation into polynucleotides.

Abstract: Propargylethoxyamino nucleosides are disclosed having the structure wherein R1 and R2 are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W1 is —H or —OH; W2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W3 is —PO4, —P2O7, —P3O10, phosphate analog, or —OH. Additionally, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Abstract: By immobilizing identical or different nucleic acids in a plurality of dot-like areas on a carrier which comprises a base material and compound carried on the base material, the compound having one or more alkylating groups, through intermediary of the alkylating group, a nucleic acid-immobilized substrate on which nucleic acids are firmly immobilized in fine dot area without reference to chain length of nucleic acids is provided, which enables efficiently introducing the nucleic acids onto the base material in a simple manner and can be produced with a simple apparatus.

Abstract: The present invention provides metal-containing purines, pyrimidines, nucleosides, nucleotides and oligonucleotides; including phosphoramidite and photolabile derivatives thereof, including methods of making and method of using same. The present invention provides a method for detection of nucleic acid sequences via electrochemical or photochemical means.

Abstract: Disclosed are compounds having two kinds of reporters that can be a donor and an acceptor for energy transfer, for example, fluorescent groups, and having a 2′, 3′-dideoxyribonucleotide residue or a 3′-deoxyribonucleotide residue. These compounds can be used as terminators for the chain terminator method. The two kinds of reporters are arranged with a distance sufficient for causing energy transfer from the donor to the acceptor. Also disclosed are methods for determining DNA sequences based on the chain terminator method wherein the chain termination reaction is performed by using the above terminators. Also disclosed are compounds having two kinds of reporters that can be a donor and an acceptor for energy transfer, which can be used as a primer or an initiator in methods for determining DNA sequences utilizing the chain terminator method, and methods for determining DNA sequences utilizing the compounds.

Abstract: The invention concerns the introduction of predetermined genetic changes in target genes of a living cell by introducing an oligodeoxynucleotide encoding the predetermined change. The oligodeoxynucleotides are effective in animal, plant and bacterial cells. Specific end modifications that greatly increase the effectiveness of the oligodeoxynucleotides in bacteria are described. Surprisingly, unmodified oligodeoxynucleotides can be as effective in mammalian cells, including in vivo hepatocytes, as the modified nucleotides and can be as effective or more effective than chimeric oligonucleotides that consist of a mixture of deoxynucleotides and 2′-O-methyl ribonucleotides.

Abstract: Biomolecule analysis using anodic oxidation of aqueous sodium 9,10-diphenylanthracene-2-sulfonate (DPAS) and 1- and 2-thianthrenecarboxylic acid (1-THCOOH and 2-THCOOH) in the presence of tri-n-propylamine (TPrA) as a coreactant in aqueous solution produces electrogenerated chemiluminescence (ECL). In addition, the cathodic reduction of DPAS in the presence of peroxydisulfate (S2O82−) as a coreactant also produces ECL in an acetonitrile (MeCN)-water solution (1:1 by volume). The oxidation of chlorpromazine (CPZ) produces an ECL emission in the absence of an added coreactant following an unprecedented “self-annihilation” mechanism.

Abstract: Compositions and methods for fluorescent detection of nucleic acids are provided. The compositions can be detected by fluorescence when hybridized to a nucleic acid containing a target sequence, but are non-fluorescent in the non-hybridized state. Alternatively, the fluorescence properties of the compositions change in a detectable manner upon hybridization to a nucleic acid containing a target sequence. Methods for synthesis and methods of use of the compositions are also provided.

Abstract: A method or detecting the presence of living or dead microorganisms and viruses in a sample comprises adding to a pre-determined volume of a sample comprising nucleic acid-containing microbe (s) and/or virus (es), known amounts of a pair of primers binding to sequences up-stream and down-stream to a universal or specific microbial and/or viral nucleic acid sequence and polymerase chain reaction (PCR) reagents, cycling the mixture to amplify the universal or specific microbial and/or viral nucleic acid sequence; adding a polynucleotide comprising a DNA internal segment that is hybridizably complementary to at least a portion of the universal or specific nucleic acid sequence; and a first and a second DNA arm segment adjoining the DNA internal segment, the first DNA arm segment ending in a 5′ terminus and the second DNA arm segment ending in a 3′ terminus, the arms segments comprising nucleotide sequences such that they are hybridizably complementary to one another.

Abstract: The present invention provides methods of screening for the presence of premalignant melanocytes in a sample from a patient. The methods comprise contacting a nucleic acid sample from a biological sample from the patient with a probe which binds selectively to a target polynucleotide sequence on a chromosomal region which is amplified in melanoma cells.

Abstract: The present invention provides methods of determining relative copy number of target nucleic acid sequences and precise mapping of chromosomal abnormalities associated with disease. The methods of the invention use target nucleic acid sequences immobilized on a solid surface, to which a sample comprising two sets of differentially labeled nucleic acid sequences are hybridized. The hybridization of the labeled nucleic acid sequences to the solid surface is then detected using standard techniques.

Abstract: A method for analyzing an objective substance, comprising reacting a labeled probe with an objective substance on a biological sample, said probe comprising a label substance of the formula (I): wherein A1 is an aromatic group, R1 is a hydrogen or —COCH2COCnF2n+1 and n is an integer of 1-6, which is bonded to a probe selected from the group consisting of nucleic acid, nucleic acid binding protein, low molecular ligand and receptor for ligand (except antibody) to give a fluorescent complex, reacting the complex with an objective substance on a biological sample and assaying fluorescence of the resultant fluorescent complex, a labeled nucleic acid probe and a labeled nucleotide. According to the method of the present invention, defects such as hindrance of fluorescence due to contaminant substance, low sensitivity and the like can be resolved, thereby enabling analysis on a tissue.

Abstract: A method for detecting the presence of a target nucleic acid sequence in a sample, said method comprising: (a) adding to a sample suspected of containing said target nucleic acid sequence, a probe specific for said target sequence and DNA duplex binding agent, said probe comprising a reactive molecule able to absorb fluorescence from or donate fluorescent energy to said DNA duplex binding agent, (b) subjecting the thus formed mixture to an amplification reaction in which target nucleic acid is amplified, (c) subjecting said sample to conditions under which the said probe hybridises to the target sequence, and (d) monitoring fluorescence from said sample. This method can be used for example to monitor amplification reactions such as PCR reactions, such that the amount of target sequence present in the sample may be determined.

Abstract: Derivatized oligonucleotides (ODNs) are coupled to a solid support in improved yield resulting in a high density of coupled oligonucleotide per surface unit of the support through a Schiff base type bond formed between an NH2 group attached either to the solid support or to the ODN and an aromatic aldehyde attached to the other of the solid support and the ODN. The preferred solid support-ODN conjugate is formed between semicarbazide groups attached to a glass surface and an aromatic aldehyde attached at either 3′, or 5′ end of an ODN or to an intermediate nucleotide of the ODN.

Abstract: A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength.

Abstract: Compositions and methods are disclosed which facilitate purification of oligomers and other compounds. The disclosed compositions are silyl compositions that can be directly coupled, or coupled through a linking group, to a compound of interest, preferably to an oligomer at the end of oligomer synthesis. The silicon atom includes between one and three sidechains that function as capture tags. In one embodiment, the capture tags are lipophilic, which allows a derivatized oligomer to be separated from failure sequences by reverse phase chromatography. In another embodiment, the capture tags are compounds with a known affinity for other compounds, which other compounds are preferably associated with a solid support to allow chromatographic separation. Examples include haptens, antibodies, and ligands. Biotin, which can bind to or interact with a streptavidin-bound solid support, is a preferred capture tag of this type.

Abstract: The present invention comprises a new class of oligonucleobases (e.g., oligonucleotides), which we call “pseudo-cyclic oligonucleotides” (PCOs). PCOs contain two oligonucleotide segments attached through their 3′-3′ or 5′-5′ ends. One of the segments (the “functional segment”) of the PCO has some functionality (e.g., an antisense oligonucleotide complementary to a target mRNA). Another segment (the “protective segment”) is complementary to the 3′- or 5′-terminal end of the functional segment (depending on the end through which it is attached to the functional segment). As a result of complementarity between the functional and protective segment segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target nucleic acids (e.g., RNA).

Abstract: A method of detecting peptide fragments of protein(s) that are differentially present in biological samples. The identity of the peptides may be determined and correlated with the protein(s) that are differentially present in the samples.

Abstract: A compound of the following formula: Ea—La—X—Lb—Eb in which each of Ea and Eb independently is a group having oxidation-reduction activity and having a conjugated system in its group; X is a divalent cyclic group; and each of La and Lb independently is a group which does not form a conjugated system in combination with the conjugated system of each of Ea and Eb and at least one of which has a site imparting water solubility to the compound or a site that is convertible into a site imparting water solubility to the compound, is favorably employable as an electroconductive threading intercalator in an electrochemical method for detecting complementary DNA fragments.

Abstract: Aryl phosphate derivatives of d4T with para-bromo substitution on the aryl group show markedly increased potency as anti-HIV agents without undesirable levels of cytotoxic activity. In particular, these derivatives are potent inhibitors of HIV reverse transcriptase. In a preferred aspect of the present invention, the phosphorus of the aryl phosphate group is further substituted with an amino acid residue that may be esterified or substituted, such as a methoxy alaninyl group.

Abstract: Spiroadamantyl dioxetanes bearing an alkoxy substituent, and an aromatic substituent of phenyl or naphthyl on the dioxetane ring can be activated to chemiluminesce if the aromatic substituent bears a moiety designated OX, wherein the X is cleaved by an enzyme with which the dioxetane is permitted to come in contact with. The T½ kinetics of the chemiluminescent reaction, as well as the signal intensity, or quantum yield of the chemiluminescent reaction, can be altered by selection of an electron-withdrawing or an electron-donating group Z, at positions on the aromatic substituent other than those adjacent the point of attachment to the dioxetane. Signal strength can further be enhanced by recognized chemiluminescent enhancers.

Abstract: Fluorescent energy transfer cassettes that allow through bond energy transfer and have a succinimidyl ester functionality suitable for affecting them to biomolecules or provided and are applied to high throughput DNA sequencing.

Abstract: The invention is directed to novel methods of making nucleosides modified with signalling moieties and polydentate ligands, particularly for use in chelating transition metal complexes to form signalling moieties such as electron transfer moieties and fluorophores.

Abstract: Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R21Z1C(O)R22R26 where R21 is a C1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z1 is either NH, sulfur or oxygen, R22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R28 includes a functional group which attaches the linker to the acceptor dye.

Abstract: The invention relates to compounds of formula (I): Fm(—A1)(—Bn)(—Wo) wherein F is a colorant-signal molecule with a maximum absorption value ranging from 600-1200 nm; A is a &bgr;-amyloid plaque binding biomolecule; B is a &bgr;-amyloid plaque binding colorant; and W is a &bgr;-amyloid plaque binding hydrophilic low-molecular structural element. The invention also describes the use of these compounds in in vivo an din vitro diagnosis of neurodegenerative diseases such as Alzheimer's disease by means of near infra-red radiation (NIR radiation) as a constrasting agent in fluoresecence and transillumination diagnosis in the NIR range. Diagnostic agents containing said componenets are also disclosed.

Abstract: The present invention provides methods for the determination of the structure of biomolecular targets, as well as the site and nature of the interaction between ligands and biomolecular targets. The present invention also provides methods for the determination of the relative affinity of a ligand for the biomolecular target it interacts with. Also provided are methods for screening ligand or combinatorial libraries of compounds against one or more than one biological target molecules. The methods of the invention also allow determination of the relative binding affinity of combinatorial and other compounds for a biomolecular target. The present invention further provides methods for the use of mass modifying tags for screening multiple biomolecular targets. In a preferred embodiment, ligands which have great specificity and affinity for molecular interaction sites on biomolecules, especially RNA can be identified.

Abstract: Novel oligonucleotide analogs of the formulae I and II in which A, B, D, R1, R2, T, U, V, W, X, Y, Z, a, b, m, m′, n and n′ have the meanings stated in the description, with valuable physical, biological and pharmacological properties, and a process for the preparation thereof are described. Application thereof relates to the use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), as probes for detecting nucleic acids and as aids in molecular biology.

Abstract: A method is described by which the association between an oligonucleotide labeled by attachment of a fluorophore and another macromolecule such as a protein or nucleic acid may be determined quantitatively in solution accurately and with high sensitivity. In the performance of this method the polarization of fluorescence of an extrinsic fluorescence probe that is covalently coupled to the oligonucleotide is determined. Changes in fluorescence polarization are related directly to the degree of association between the labeled oligonucleotide and another macromolecule and may be used to quantify the association. Because of its high sensitivity and accuracy, this method may be used to make reliable quantitative measurements of very small amounts of complexes formed between labeled oligonucleotides and proteins, nucleic acids or other macromolecules. The method also allows the accurate calculation of important biochemical parameters such as dissociation constants.

Abstract: Dibenzorhodamine compounds having the structure are disclosed, including nitrogen and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH2OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Abstract: The invention relates to oligonucleotides labeled with an energy transfer acceptor useful in conjunction with fluorescent nucleic acid stains. The resulting oligonucleotides are useful for decreasing background fluorescence during amplification assays and in ligation assays, and for detecting hybridization.

Abstract: Tetrahydrofuranyl compounds are provided that are functionalized to include pendant conjugate groups, and which are useful in diagnosis assays and as research reagents. Novel intermediates for the synthesis of the compounds are also provided.

Abstract: Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI3 group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides.

Abstract: A technique for immobilizing biological molecules, in particular nucleic acid strands, is described. Biological molecules immobilized at surfaces can be used in electron-transfer detection techniques in which a binding partner of a biological molecule is brought into proximity of the surface-immobilized biological molecule, an electrical potential created between the two biologically-binding species, and electron transfer through the species determined. Another technique involves immobilizing a bioligical molecule such as a protein, DNA, etc. at a surface via a self-assembled monolayer, affecting the biological molecule via, for example, biological binding, inducing a change in conformation via a prion, etc., and detecting an electronic property change in the molecule via a change in inpedence associated with an electronic circuit addressed by the biological molecule. These technique facilitates combinatorial array detection articles.

Abstract: Methods and compositions are provided for forming complexes intracellularly between dsDNA and oligomers of heterocycles, aliphatic amino acids, particularly omega-amino acids, and a polar end group. By appropriate choice of target sequences and composition of the oligomers, complexes are obtained with low dissociation constants. The formation of complexes can be used for modifying the phenotype of cells, either prokaryotic or eukaryotic, for research and therapy.

Abstract: A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure wherein Y1 and Y2 are individually hydroxyl, amino, imminium, or oxygen, R1-R8 are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R9 is acetylene, alkane, alkene, cyano, substituted phenyl, and combinations thereof. The invention further includes novel intermediate compounds useful for the synthesis of asymmetric benzoxanthene compounds having the general structure where substituents R3-R7 correspond to like-referenced substituents in the structure of described above, and Y2 is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates.

Abstract: The invention concerns a novel class of 3′-modified, pro-fluorescent nucleotides. The invention also pertains to methods for using such nucleotides.

Abstract: Nucleic acid labeling compounds containing heterocyclic derivatives are disclosed. The heterocyclic derivative containing compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

Abstract: Novel fluorescent labeling techniques and fluorescent labels are provided, employing high affinity non-covalently binding and intercalating fluorescent dyes and dsDNA. The dyes find application to provide highly sensitive labeling of nucleic acids in electrophoretic gels and as pre-prepared labels for binding to a wide variety of specific binding pair members. The DNA-dye fluorescer complex can be used for labels in diagnostic assays, detection of specific nucleic acid sequences, and the like.

Abstract: A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or Tm. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the Tm has been found to be significantly lower than the Tm for a perfectly match probe/stand duplex.

Abstract: The invention concerns the introduction of predetermined genetic changes in target genes of a living cell by introducing an oligodeoxynucleotide encoding the predetermined change. The oligodeoxynucleotides are effective in mammalian, avian, plant and bacterial cells. Specific end modifications that greatly increase the effectiveness of the oligodeoxynucleotides in bacteria are described. Surprisingly, unmodified oligodeoxynucleotides can be as effective in mammaliancells, including in vivo hepatocytes, as the modified nucleotides and can be as effective or more effective than chimeric oligonucleotides that consist of a mixture of deoxynucleotides and 2′-O-methyl ribonucleotides.

Abstract: A method for the detection of diagnostic base sequences in a sample nucleic acid comprises contacting the sample in the presence of appropriate nucleoside triphosphates and an agent for polymerization thereof with a diagnostic primer for the diagnostic base sequence, the primer having a tail sequence comprising a tag region and a detector region such that an extension product of the primer is synthesized when the corresponding base sequence is present in the sample, and any extension product of the diagnostic primer acting as a template for extension of a further primer which hybridizes to a locus at a distance from the diagnostic base sequence. The sample is contacted with a tag primer which selectively hybridizes to the complement of the tag sequence in an extension product of the further primer and is extended, and the presence or absence of the diagnostic base sequence is detected by reference to the detector region in the further primer extension product.

Abstract: A method of analysing a nucleic acid by mass spectrometry comprising the steps of: (1) preparing a nucleic acid molecule comprising a negatively charged non-phosphate sugar-sugar linkage; (2) eliminating the charge from all, or up to all but ten, of the sugar-sugar linkages of the said nucleic acid molecule; (3) introducing the said nucleic acid molecule in which the charge has been wholly or partly eliminated as said into a mass spectrometer; and (4) determining the mass of the said nucleic acid molecule. Preferably, the nucleic acid has no or one charge. A method of preparing a nucleic acid molecule containing no or up to ten negative charges and no or up to ten positive charges comprising the steps of (1) synthesizing a nucleic acid with a phosphorothioate linkage or a phosphoroselenoate linkage between sugar residues, and (2) reacting the said nucleic acid with an alkylating agent so as to eliminate the charge on the said phosphorothioate linkage or said phosphoroselenoate linkage.

Abstract: The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

Abstract: A process for aligning a macromolecule (macromolecules) on the surface S of a support is characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on the said surface S, the said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A. The subject of the present invention is also a process for detecting, measuring the intramolecular distance of, separating and/or assaying a macromolecule in a sample in which a process of alignment according to the invention is used.

Abstract: A stable complex, we refer to as a PD-Loop, between double stranded nucleic acid and a nucleobase polymer is assembled with the aid of strand invading peptide nucleic acid (PNA). The PD-Loop can be used in the detection, analysis, quantitation and even in the affinity capture of the duplex nucleic acid. Alternatively, the PD-Loop can be used to initiate polymerase extension of a primer to thereby facilitate sequencing of the double stranded nucleic acid even in the presence of large excesses of unrelated double stranded nucleic acid. As an additional feature, the PD-Loop can also be used to generate a construct comprised of a double stranded nucleic acid through which is threaded a single stranded closed circular nucleic acid wherein the closed circular nucleic acid can be used in a signal amplification methodology.