Mutation detection assay

A method of sample analysis comprising:

• 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 the wild type copies of the genomic locus, to produce an amplified sample;
• wherein:
• i. the amplifying is performed 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;
• b) detecting the presence of the product in the amplified sample using a flap assay that employs:
• i. an invasive oligonucleotide distinct from the first primer, the invasive oligonucleotide comprising a 3′ terminal nucleotide that base pairs with the point mutation;
• and
• ii. a flap oligonucleotide that comprises a nucleotide that base pairs with the point mutation.

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 nucleic acid sample;
• wherein the first primer:
• i. comprises a 3′ terminal nucleotide that base pairs with a point mutation in the genomic locus;
• and
• ii. comprises a nucleotide sequence that is fully complementary to a sequence in the locus except for a single base mismatch within 6 bases of the 3′ terminal nucleotide;
• b) flap assay reagents comprising an invasive oligonucleotide distinct from the first primer, wherein the invasive oligonucleotide comprises a 3′ terminal nucleotide that base pairs with the point mutation, a flap endonuclease, a FRET cassette, and a flap oligonucleotide that comprises a nucleotide that base pairs with the point mutation;
• c) the nucleic acid sample, wherein the nucleic acid sample comprises both wild type copies of the genomic locus and mutant copies of the genomic locus that have a point mutation relative to the wild type copies of the genomic locus;
• wherein the reaction mixture is characterized in that it can amplify and detect the presence of the mutant copies of the genomic locus in the sample.

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 the first primer comprises a 3′ terminal nucleotide that base pairs with a point mutation in the locus in human genomic DNA 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) flap assay reagents that include a flap endonuclease, a FRET cassette and a flap oligonucleotide that comprises a nucleotide that base pairs with the point mutation,
• wherein the kit comprises an invasive oligonucleotide that is distinct from the first primer wherein the invasive oligonucleotide comprises a 3′ terminal nucleotide that base pairs with the point mutation, and wherein the PCR reagents and flap assay reagents are selected to effect amplification and detection of mutant copies of the locus when they are combined with a nucleic acid sample that comprises wild type and mutant copies of the genomic locus and subjected to thermocycling.