Multiplexed KRAS mutation detection assay
Provided herein is reagent mixture comprising multiplexed amplification reagents and flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T, 35A, 35C, 35T or 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided.
1. A composition comprising:
at least seven different primers that each comprise, at their 3′ end, at least 12 contiguous nucleotides starting from the 3′ end of a sequence selected from SEQ ID NOs: 1-7.
2. The composition of claim 1 , wherein the composition further comprises a thermostable polymerase.
3. The composition of claim 1 , wherein the composition further comprises nucleotides.
4. The composition of claim 1 , wherein the primers are each complementary to at least 16 nucleotides in the human KRAS gene.
5. The composition of claim 1 , wherein the primers each comprise a sequence selected from SEQ ID NOS: 1-7.
6. The composition of claim 1 , further comprising a reverse primer that hybridizes to the human KRAS gene.
7. The composition of claim 1 , further comprising flap assay reagents that comprise a flap endonuclease, a FRET cassette that produces a fluorescent signal when cleaved, and at least seven different flap oligonucleotides.
8. The composition of claim 7 , wherein the flap oligonucleotides each base pair with 10 to 14 contiguous nucleotides of the human KRAS gene.
9. The composition of claim 8 , wherein the flap oligonucleotides each comprise at their 3′ ends at least 11 contiguous nucleotides from the 3′ end of a sequence as set forth in SEQ ID NOs: 11-17.
10. The composition of claim 1 , further comprising a nucleic acid extracted from a sample.
11. The composition of claim 10 , wherein the nucleic acid comprises at least a 100-fold excess of wild type copies of the KRAS gene relative to mutant copies of the KRAS gene that contain a point mutation selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A.
12. The composition of claim 10 , wherein the sample is obtained from a human.
13. The composition of claim 10 , wherein the sample is a stool sample.
14. The composition of claim 10 , wherein the sample is a tissue or biopsy sample.
15. The composition of claim 1 , wherein the composition comprises:
a) amplification reagents comprising a thermostable polymerase, nucleotides, said at least seven different primers, and a reverse primer, wherein each of the seven different 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 a point mutation selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A; and
b) flap assay reagents comprising a flap endonuclease, a first FRET cassette that produces a fluorescent signal when cleaved, said at least seven different primers, and a corresponding set of at least seven different flap oligonucleotides that each comprise a nucleotide that base pairs with one of the point mutations.
16. The composition of claim 15 , wherein the seven different flap oligonucleotides comprise at their 3′ ends at least 11 contiguous nucleotides from the 3′ end of a sequence as set forth in SEQ ID NOs: 11-17.