Methylation assay

摘要

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.

主权项

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 in a reaction mixture 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 wherein said amplifying comprises subjecting said reaction mixture to a first set of 5-15 cycles of:
1. a first temperature of at least 90° C.;2. a second temperature in the range of 60° C. to 75° C.;3. a third temperature in the range of 65° C. to 75° C.; and c) detecting the presence of amplified methylated copies of said genomic locus in said amplified sample without adding further reagents to the reaction mixture 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; wherein said detecting comprises subjecting the amplified sample to a second set of 20-50 cycles of:
4. a fourth temperature of at least 90° C.;5. a fifth temperature that is at least 10° C. lower than said second temperature;6. a sixth temperature in the range of 65° C. to 75° C.; andwherein said first primer of step b) is used as said invasive oligonucleotide in said flap assay of step c) and, in addition to having a 3′ terminal G or C nucleotide that corresponds to a methylated cytosine in said genomic locus, said first primer comprises an internal G or C nucleotide at a position that corresponds to a second methylated cytosine in said genomic locus.