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.

What is claimed is: 
     
         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 . 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 optionally 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, a flap oligonucleotide and, if said first primer does not contain said 3′ terminal nucleotide, an invasive oligonucleotide distinct from said first primer, wherein i. said invasive oligonucleotide has a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in said genomic locus and ii. said a flap oligonucleotide comprises a G or C nucleotide at a position that corresponds to said methylated cytosine; 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 methylated 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.   
     
     
         18 . The reaction mixture of  claim 17 , 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. 
     
     
         19 . The reaction mixture of  claim 17 , 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. 
     
     
         20 . The reaction mixture of  claim 17 , wherein said first and second primers both hybridize to a methylated sequence in said genomic locus. 
     
     
         21 . The reaction mixture of  claim 17 , wherein:
 i. said first primer contains a 3′ terminal nucleotide that base pairs with a methylated cytosine in said methylated sequence; and   ii. said flap assay reagents do not comprise said invasive oligonucleotide.   
     
     
         22 . The reaction mixture of  claim 17 , wherein:
 i. said first primer does not contain a 3′ terminal nucleotide that base pairs with a methylated cytosine in said methylated sequence; and   ii. said flap assay reagents comprise said invasive oligonucleotide.   
     
     
         23 . The reaction mixture of  claim 17 , wherein:
 the reaction mixture comprises a first primer and an invasive oligonucleotide that each contain a 3′ terminal nucleotide that base pairs with a methylated cytosine in said genomic locus, wherein the 3′ terminal nucleotide of said first primer and the 3′ terminal nucleotide of said invasive oligonucleotide base pair with different methylated sites in said genomic locus.   
     
     
         24 . A kit comprising:
 a) PCR reagents that include a first primer and a second primer, where the first primer hybridizes to a methylated sequence in the genomic locus and optionally contains a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in the methylated sequence; and   b) flap assay reagents comprising a flap endonuclease, a FRET cassette and, if the first primer does not contain said 3′ terminal nucleotide, an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.   
     
     
         25 . The kit of  claim 24 , further comprising instructions for performing the method of  claim 1 .