Mutation detection assay

摘要

A method of sample analysis is provided. In certain embodiments, the method involves: a) amplifying a product from a sample that comprises both wild type copies of a genomic locus and mutant copies of the genomic locus that have a point mutation relative to said wild type copies of the genomic locus, to produce an amplified sample, where: i. the amplifying is done using a first primer and a second primer; and ii. the first primer comprises a 3′ terminal nucleotide that base pairs with the point mutation and also comprises a nucleotide sequence that is fully complementary to a sequence in the locus with the exception of a single base mismatch within 6 bases of the 3′ terminal nucleotide; and b) detecting the presence of said product in said amplified sample using a flap assay that employs an invasive oligonucleotide. A kit for performing the method is also provided.

主权项

1. A kit for detecting mutant copies of a locus in human genomic DNA, comprising:
a) PCR reagents that include a first primer and a second primer, wherein said first primer comprises a 3′ terminal nucleotide that base pairs with a point mutation in said locus in human genomic DNA and also comprises a nucleotide sequence that is fully complementary to a sequence in said locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide; and b) flap assay reagents that include a flap endonuclease, a FRET cassette and a flap oligonucleotide that comprises a nucleotide that base pairs with said point mutation, wherein the kit does not comprise an invasive oligonucleotide that is distinct from said first primer and wherein the PCR reagents and flap assay reagents are selected to effect amplification and detection of mutant copies of said locus when they are combined with a nucleic acid sample that comprises wild type and mutant copies of said genomic locus and subjected to thermocycling.