Methylation assay

A reaction mixture comprising:

• a) amplification reagents comprising a thermostable polymerase, nucleotides, a first primer and a second primer for amplifying a target genomic locus from a treated nucleic acid sample;
• wherein:
• i. said first primer hybridizes to a methylated sequence in said locus and contains a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in said genomic locus;
• and
• ii. said reagents preferentially amplify methylated copies of said genomic locus, to produce an amplified sample;
• b) flap assay reagents comprising a flap endonuclease, a FRET cassette, and a flap oligonucleotide that comprises a G or C nucleotide at a position that corresponds to said methylated cytosine and wherein an invasive oligonucleotide distinct from the first primer is not included and wherein the first primer serves as an invasive oligonucleotide;
• and
• c) said treated nucleic acid sample, wherein said treated nucleic acid sample is made by treating an initial nucleic acid sample comprising both methylated copies and unmethylated copies of said genomic locus with an agent that modifies unmethylated cytosine to uracil;
• wherein said reaction mixture is characterized in that it can amplify and detect the presence of methylated copies of said genomic locus in said sample.