Abstract: Provided are nucleic acid detection methods wherein targeted primer extension or products or amplification products incorporate a probe-anchoring modification introduced using a primer incorporating a probe-anchoring primer modification, wherein oligonucleotide detection probes incorporate a probe-anchoring probe modification, the primers and probes designed to place the binding site of the oligonucleotide probe proximate to the probe-anchoring primer modification in the detected target sequence. The probe-anchoring modifications of the probe and the primer-extension product form a stabilized complex comprising a detectible duplex of the probe with the detected target sequence. In certain aspects, the probe-anchoring modified primer also incorporates a probe-directing sequence. The methods allow use of exceptionally short oligonucleotide probes (e.g.

Abstract: Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, the methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences.

Abstract: Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5?-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing the oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3?- or 5?-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid.

Abstract: Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, said methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences.

Abstract: Aspects of the invention provide novel and surprisingly effective methods for the detection of nucleic acids, comprising nucleic acid amplification using base-modified deoxynucleoside 5?-triphosphates (dNTPs). Particular aspects relate to methods for enhancing hybridization properties of oligonucleotide primers and probes in assays detecting nucleic acids, comprise amplifying target DNAs in presence of base-modified duplex-stabilizing deoxyribonucleoside 5?-triphosphates to provide for modified target DNAs, and thereby considerably improving performance of the detection assays. The disclosed methods allow for increasing of the reaction temperature in PCR-based detection systems or, alternatively, reducing the length of the oligonucleotide primers and probes. Certain aspects relates to improvement of real time PCR assays, wherein nucleic acids of interest are detected as the reaction proceeds using fluorescent agents or oligonucleotide FRET probes.

Abstract: Provided are nucleic acid detection methods wherein targeted primer extension or products or amplification products incorporate a probe-anchoring modification introduced using a primer incorporating a probe-anchoring primer modification, wherein oligonucleotide detection probes incorporate a probe-anchoring probe modification, the primers and probes designed to place the binding site of the oligonucleotide probe proximate to the probe-anchoring primer modification in the detected target sequence. The probe-anchoring modifications of the probe and the primer-extension product form a stabilized complex comprising a detectible duplex of the probe with the detected target sequence. In certain aspects, the probe-anchoring modified primer also incorporates a probe-directing sequence. The methods allow use of exceptionally short oligonucleotide probes (e.g.

Abstract: Particular aspects provide nucleic acid amplification and detection methods comprising: providing a reaction mixture containing a target nucleic acid with an amplifiable target sequence, forward and reverse external nick-directing primers (ND-primers), at least one internal ND-primer, a strand-displacing DNA polymerase, a nick-directing endonuclease for strand-specific cleavage of ND-primer-extension products, and deoxynucleoside 5?-triphosphates; and incubating the reaction mixture with reagents, and under conditions suitable to provide for amplification of the amplifiable target sequence, wherein the amplification comprises primer extension, by least one internal ND-primer, of an external ND-primer extension product comprising the amplifiable target sequence or a portion thereof but lacking the respective external ND-primer sequence or a portion thereof.

Abstract: Particular aspects provide nucleic acid detection methods comprising contacting a test sample having a nucleic acid target sequence with at least one endo-V-cleavable oligonucleotide probe complementary to the target sequence in the presence of an endonuclease V, incubating the reaction mixture under conditions suitable to support hybridization of the endo-V-cleavable oligonucleotide probe with the target nucleic acid and endonuclease V-mediated cleavage of the target-hybridized probe, and detecting at least one endonuclease V-mediated cleavage product of the target-hybridized probe wherein the presence of the cleavage products is indicative of the presence of the target nucleic acid sequence in the sample. Particular aspects comprise amplification of the target nucleic acid sequence before and/or during the incubating and/or detecting, wherein detecting is post-amplification and/or real-time. Additional aspects provide suitable kits.

Abstract: Aspects of the invention provide novel and surprisingly effective methods for the detection of nucleic acids, comprising nucleic acid amplification using base-modified deoxynucleoside 5?-triphosphates (dNTPs). Particular aspects relate to methods for enhancing hybridization properties of oligonucleotide primers and probes in assays detecting nucleic acids, comprise amplifying target DNAs in presence of base-modified duplex-stabilizing deoxyribonucleoside 5?-triphosphates to provide for modified target DNAs, and thereby considerably improving performance of the detection assays. The disclosed methods allow for increasing of the reaction temperature in PCR-based detection systems or, alternatively, reducing the length of the oligonucleotide primers and probes. Certain aspects relates to improvement of real time PCR assays, wherein nucleic acids of interest are detected as the reaction proceeds using fluorescent agents or oligonucleotide FRET probes.

Abstract: Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5?-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing said oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3?- or 5?-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid.

Abstract: Particular aspects provide nucleic acid amplification and detection methods comprising: providing a reaction mixture containing a target nucleic acid with an amplifiable target sequence, forward and reverse external nick-directing primers (ND-primers), at least one internal ND-primer, a strand-displacing DNA polymerase, a nick-directing endonuclease for strand-specific cleavage of ND-primer-extension products, and deoxynucleoside 5?-triphosphates; and incubating the reaction mixture with reagents, and under conditions suitable to provide for amplification of the amplifiable target sequence, wherein the amplification comprises primer extension, by least one internal ND-primer, of an external ND-primer extension product comprising the amplifiable target sequence or a portion thereof but lacking the respective external ND-primer sequence or a portion thereof.

Abstract: The present invention provides a novel method for detection and/or genotyping of nucleic acids that utilizes the specificity of an AP endonuclease. In addition, the present invention provides a novel method for nucleic acid amplification.

Abstract: Oligonucleotide probes/conjugates are provided along with method for their use in assays to monitor amplification wherein the signal produced does not rely on 5? nuclease digestion.

Abstract: Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Abstract: Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Abstract: The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.

Abstract: Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

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.