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 reaction mixture comprising:
(a) amplification reagents comprising a thermostable polymerase, nucleotides, a first primer and a second primer, wherein the first primer and the second primer amplify a target genomic locus from a nucleic acid sample comprising human genomic DNA and said first primer comprises:
(i) a 3′ terminal nucleotide that base pairs with a point mutation in said target genomic locus; and(ii) a nucleotide sequence that is fully complementary to a sequence in said target genomic locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide; (b) assay reagents comprising a probe that contains a flap, a hairpin oligonucleotide that is capable of hybridizing to the flap after said flap is released from the probe and that comprises a terminal fluorophore moiety and a quencher moiety, and a flap endonuclease, wherein said fluorophore is released from said hairpin oligonucleotide by said flap endonuclease during the amplification of said genomic locus; and (c) said nucleic acid sample, wherein said nucleic acid sample comprises both wild type copies of said target genomic locus and mutant copies of said target genomic locus and wherein the mutant copies of said target genomic locus have said point mutation of step (a)(i),
wherein said reaction mixture is characterized in that upon thermocycling of the reaction mixture the mutant copies of said genomic locus are amplified and the terminal fluorophore is cleaved from the hairpin oligonucleotide, thereby changing the fluorophore from a quenched state to an unquenched state to produce a fluorescent signal that allows the mutant copies of said genomic locus to be detected by a fluorometer.