Methylation assay

Abstract

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.

1 . A method for detecting a methylated genomic locus, comprising:
 a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies unmethylated cytosine to uracil to produce a treated nucleic acid;   b) amplifying a product from said treated nucleic acid using a first primer and a second primer, wherein said first primer hybridizes to a methylated sequence in said locus and said amplifying preferentially amplifies said methylated copies of said genomic locus, to produce an amplified sample; and   c) detecting the presence of amplified methylated copies of said genomic locus in said amplified sample using a flap assay that employs: i. an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in said genomic locus and ii. a flap oligonucleotide that comprises a G or C nucleotide at a position that corresponds to said methylated cytosine in said genomic locus.   
     
     
         2 . The method of  claim 1 , wherein said flap probe comprises an internal G or C nucleotide at a position that corresponds to a second methylated cytosine in said genomic locus. 
     
     
         3 . The method of  claim 1 , wherein said first primer comprises an internal G or C nucleotide at a position that corresponds to a second methylated cytosine in said genomic locus. 
     
     
         4 . The method of  claim 1 , wherein said first and second primers both bind to sites that contain methylcytosines in said genomic locus. 
     
     
         5 . The method of  claim 1 , wherein said first primer is used as said invasive oligonucleotide in said flap assay. 
     
     
         6 . The method of  claim 1 , wherein said nucleic acid sample contains at least 100 times more unmethylated copies of said genomic locus than methylated copies of said genomic locus. 
     
     
         7 . The method of  claim 1 , further comprising normalizing the amount of said amplified methylated copies of said genomic locus in said amplified sample relative to the amount of a control nucleic acid present in said nucleic acid sample, thereby determining the amount of methylated copies of said genomic locus in said nucleic acid sample. 
     
     
         8 . The method of  claim 7 , wherein said control nucleic acid is a locus different from said genomic locus. 
     
     
         9 . The method of  claim 7 , wherein said control nucleic acid is detected using a flap assay that employs an invasive oligonucleotide having a 3′ terminal nucleotide that base pairs with an A or T residue at the site of said methylated cytosine, thereby detecting the presence of unmethlyated copies of said genomic locus. 
     
     
         10 . The method of  claim 7 , wherein the said flap assay employs first flap assay reagents that include a first invasive oligonucleotide, a first flap probe having a first flap and a first FRET cassette, and wherein said control nucleic acid is detected using second flap assay reagents that include a second invasive oligonucleotide, a second flap probe having a second flap and a second FRET cassette that produces a signal that is distinguishable from the first FRET cassette, wherein the first and second flap reagents are in same reaction mix. 
     
     
         11 . The method of  claim 1 , wherein methylation of said locus is cancer-related. 
     
     
         12 . The method of  claim 1 , wherein said locus is that of BMP3, TFPI1, NDRG4, Septin 9, TFPI2, or Vimentin. 
     
     
         13 . The method of  claim 1 , wherein said sample is obtained from a human. 
     
     
         14 . The method of  claim 13 , wherein said sample is stool. 
     
     
         15 . The method of  claim 1 , wherein said amplifying and detecting steps are done using a reaction mix that contains both PCR reagents and flap reagents, and no additional reagents are added to said reaction mix between said amplifying and detecting steps. 
     
     
         16 . The method of  claim 15 , wherein said reaction mix further comprises PCR reagents and flap reagents for amplifying and detecting a second genomic locus. 
     
     
         17 - 25 . (canceled)