5′ hairpin oligonucleotide substrates for reversible repression, e.g., by temperature shift, of cleavage by flap endonucleases, and methods using 5′ hairpin oligonucleotides.

5′ hairpin oligonucleotide substrates for reversible repression, e.g., by temperature shift, of cleavage by flap endonucleases, and methods using 5′ hairpin oligonucleotides.

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

Embodiments of the methods and compositions provided herein relate to the selective cleavage of target nucleic acids. Some embodiments include recombinase-mediated selective cleavage of target nucleic acids with single-stranded nucleic acid probes and a recombinase. Some embodiments also include the enrichment of non-target nucleic acids in a sample by selective cleavage of target nucleic acids in the sample, and removal of the cleaved target nucleic acids from the sample.

Embodiments of the methods and compositions provided herein relate to the selective cleavage of target nucleic acids. Some embodiments include recombinase-mediated selective cleavage of target nucleic acids with single-stranded nucleic acid probes and a recombinase. Some embodiments also include the enrichment of non-target nucleic acids in a sample by selective cleavage of target nucleic acids in the sample, and removal of the cleaved target nucleic acids from the sample.

A method for detecting a methylated genomic locus is provided. In certain embodiments, the method comprises: a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies cytosine to uracil to produce a treated nucleic acid; b) amplifying a product from the treated nucleic acid using a first primer and a second primer, wherein the first primer hybridizes to a site in the locus that contain methylcytosines and the amplifying preferentially amplifies the methylated copies of the genomic locus, to produce an amplified sample; and c) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.

A method for detecting a methylated genomic locus is provided. In certain embodiments, the method comprises: a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies cytosine to uracil to produce a treated nucleic acid; b) amplifying a product from the treated nucleic acid using a first primer and a second primer, wherein the first primer hybridizes to a site in the locus that contain methylcytosines and the amplifying preferentially amplifies the methylated copies of the genomic locus, to produce an amplified sample; and c) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.

The present invention is generally drawn to a novel method for determining a SNP (single nucleotide polymorphism) genotype using a PTO-SNV (Probing and Tagging Oligonucleotide for Single Nucleotide Variation). The present invention provides novel protocols for SNP genotyping in which only one allele-specific oligonucleotide permits in a SNP genotyping reaction to determine whether a target nucleic acid sequence to be analyzed is homozygous or heterozygous for the SNP allele of interest or has no SNP allele of interest.

The present invention is generally drawn to a novel method for determining a SNP (single nucleotide polymorphism) genotype using a PTO-SNV (Probing and Tagging Oligonucleotide for Single Nucleotide Variation). The present invention provides novel protocols for SNP genotyping in which only one allele-specific oligonucleotide permits in a SNP genotyping reaction to determine whether a target nucleic acid sequence to be analyzed is homozygous or heterozygous for the SNP allele of interest or has no SNP allele of interest.