Methylation assay
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 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.
2. The reaction mixture of claim 1 , wherein said flap oligonucleotide comprises an internal G or C nucleotide at a position that corresponds to a second methylated cytosine in said genomic locus.
3. The reaction mixture 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 reaction mixture of claim 1 , wherein said first and second primers both hybridize to a methylated sequence in said genomic locus.
5. 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 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 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.
6. The reaction mixture of claim 1 , wherein the target genomic locus is Vimentin.
7. The reaction mixture of claim 6 , wherein the methylated sequence in said locus comprises the nucleic acid sequence of SEQ ID NO:12.
8. The reaction mixture of claim 6 , wherein the first primer comprises the sequence of SEQ ID NO:13.
9. The reaction mixture of claim 6 , wherein the second primer comprises the sequence of SEQ ID NO:14.
10. The reaction mixture of claim 6 , wherein the flap oligonucleotide comprises the sequence of SEQ ID NO:15.
11. The reaction mixture of claim 1 , wherein
the first primer comprises the sequence of SEQ ID NO:13,
the second primer comprises the sequence of SEQ ID NO:14, and
the flap oligonucleotide comprises the sequence of SEQ ID NO:15.
12. The kit of claim 5 , wherein the methylated sequence is a methylated sequence to which the first primer hybridizes is a methylated sequence of Vimentin.
13. The kit of claim 12 , wherein the methylated sequence in said locus comprises the nucleic acid sequence of SEQ ID NO:12.
14. The kit of claim 12 , the first primer comprises the sequence of SEQ ID NO:13.
15. The kit of claim 12 , wherein the second primer comprises the sequence of SEQ ID NO:14.
16. The kit of claim 12 , wherein
the first primer comprises the sequence of SEQ ID NO:13, and
the second primer comprises the sequence of SEQ ID NO:14.