Multiplexed kras mutation detection assay

A reagent mixture comprising:

• a) amplification reagents comprising a thermostable polymerase, nucleotides, a set of at least seven forward primers, and a reverse primer, wherein:
• i. the 3′ terminal nucleotide of each forward primer of said set base pairs with a different point mutation in the KRAS gene relative to other forward primers in said set, wherein said point mutation is selected from the following point mutations:
• 34A, 34C, 34T, 35A, 35C, 35T and 38A;
• ii. each of said forward primers comprises a nucleotide sequence that is fully complementary to a sequence in said KRAS gene with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide;
• and
• iii. each of said forward primers, in combination with said reverse primer, selectively amplifies a different allele of a KRAS gene, wherein the allele that is amplified is defined by the point mutation to which said 3′ terminal nucleotide base pairs;
• and
• b) flap assay reagents comprising a flap endonuclease, a first FRET cassette that produces a fluorescent signal when cleaved, said set of at least seven forward primers, and a corresponding set of at least seven different flap oligonucleotides that each comprise a nucleotide that base pairs with one of said point mutations;
• wherein said reagent mixture is characterized in that, when said reagent mixture combined with a nucleic acid sample that comprises at least a 1,000-fold excess of wild type copies of said KRAS gene relative to mutant copies of said KRAS gene that contain one of said point mutations and thermocycled, said reagent mixture can amplify and detect the presence of said mutant copies of the KRAS gene in said sample.