Mutation detection assay
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.
1. A method of sample analysis, comprising:
(a) subjecting a reaction mix comprising:
a sample that comprises both wild type copies of a genomic locus and mutant copies of said genomic locus that have a point mutation relative to said wild type copies of the genomic locus, a thermostable polymerase, a FLAP endonuclease, nucleotides, a flap oligonucleotide, and a FRET cassette that comprises quencher and a fluorophore, 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,
to the following thermocycling conditions:
i. an initial pre-incubation;
ii. a first set of 5 to 15 cycles each comprising a first temperature of over 90° C. and a second temperature in the range of 65° C. to 75° C., wherein all of the cycles are the same; followed by:
iii. a second set of 20 to 50 cycles each comprising a first temperature of over 90° C. and a second temperature of 55° C. or below; and
(b) measuring a fluorescent signal in each of the second set of at least 20 to 50 thermocycles, wherein the fluorescent signal is generated by cleavage of the FRET cassette by the FLAP endonuclease and indicates the presence of a mutant locus in the sample.
2. The method of claim 1 , wherein said sample contains at least 100 times more wild type copies of said genomic locus than mutant copies of said genomic locus.
3. The method of claim 1 , wherein said sample contains at least 1,000 times more wild type copies of said genomic locus than mutant copies of said genomic locus.
4. The method of claim 1 , wherein the point mutation of said genomic locus is associated with cancer.
5. The method of claim 1 , wherein said genomic locus is the KRAS gene.
6. The method of claim 1 , wherein said genomic locus is the BRAF gene.
7. The method of claim 1 , wherein said sample is obtained from a human.
8. The method of claim 6 , wherein sample is stool.
9. The method of claim 7 , further comprising making a diagnosis of colon cancer or adenoma based on whether mutant copies of said genomic locus are identified in said stool.
10. The method of claim 1 , wherein said mismatch in said first primer is at position −1, position −2, position −3, position −4 or position −5 relative to said terminal nucleotide.
11. The method of claim 1 , wherein the cycles of the second set of 20 to 50 cycles each comprises a temperature that is in the range of 50° C. to 55° C.
12. The method of claim 1 , wherein the initial pre-incubation of (a) activates a thermostable polymerase.
13. The method of claim 1 , wherein the measuring step of (b) comprises measuring cleavage of a fluorophore from an oligonucleotide in each of the second set of 20 to 50 cycles.
14. The method of claim 1 , wherein the method further comprises graphing the amount of fluorescent signal that occurs at each of the 20-50 cycles, thereby providing an estimate of the abundance of the mutant locus in the reaction mix.
15. The method of claim 1 , wherein step (b) comprises measuring two fluorescent signals in each of the second set of at least 20 to 50 cycles, wherein the fluorescent signals respectively indicate the presence of two different mutations.
16. The method of claim 1 , wherein steps (a) and (b) of the method are done using a reaction mixture that contains both PCR reagents and flap reagents, and no additional reagents are added to said reaction mixture between said amplifying and detecting steps.
17. The method of claim 1 , wherein the thermocycling conditions comprise:
i. an initial pre-incubation;
ii. a first set of 5 to 15 cycles each comprising a first temperature of over 90° C., a second temperature in the range of 65° C. to 75° C., and a third temperature in the range of 65° C. to 75° C., wherein all of the cycles are the same; followed by:
iii. a second set of 20 to 50 cycles each comprising a first temperature of over 90° C., a second temperature of 55° C. or below and a third temperature in the range of 65° C. to 75° C.