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) amplifying a product from 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, to produce an amplified sample;
wherein:
i. said amplifying is done using a first primer and a second primer; and
ii. said first primer comprises a 3′ terminal nucleotide that base pairs with said point mutation and also comprises a nucleotide sequence that is fully complementary to a sequence in said locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide;
b) detecting the presence of said product in said amplified sample using a flap assay that employs:
i. an invasive oligonucleotide having a 3′ terminal nucleotide that base pairs with said point mutation and
ii. a flap oligonucleotide that comprises a nucleotide that base pairs with said point mutation.
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 , further comprising normalizing the amount of said product in said amplified sample relative to the amount of a control nucleic acid present in said sample, thereby determining the amount of said mutant copies in said sample.
4. The method of claim 3 , wherein said control nucleic acid is a locus different from said genomic locus.
5. The method of claim 3 , wherein said control nucleic acid is detected using a flap assay that employs an invasive oligonucleotide having a 3′ terminal nucleotide that base pairs with said wild type copies of said genomic locus at the site of said point mutation, thereby detecting the presence of wild type copies of said genomic locus in said sample.
6. The method of claim 3 , wherein said flap assay employs first flap assay reagents that include a first invasive oligonucleotide, a first flap probe having a first flap and a first FRET cassette, and wherein said control nucleic acid is detected using second flap assay regents that include a second invasive oligonucleotide, a second flap probe having a second flap and a second FRET cassette that produces a signal that is distinguishable from the first FRET cassette, wherein the first and second flap reagents are in same reaction mixture.
7. The method of claim 1 , wherein mutation of said genomic locus is associated with cancer.
8. The method of claim 7 , wherein said genomic locus is the KRAS gene.
9. The method of claim 7 , wherein said genomic locus is the BRAF gene.
10. The method of claim 1 , wherein said sample is obtained from a human.
11. The method of claim 10 , wherein sample is stool.
12. The method of claim 11 , further comprising making a diagnosis of colon cancer or adenoma based on whether mutant copies of said genomic locus are identified in said stool.
13. 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.
14. The method of claim 1 , wherein said amplifying and detecting steps 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.
15. The method of claim 14 , wherein said reaction mixture further comprises PCR reagents and flap reagents for amplifying and detecting a second genomic locus.
16. The method of claim 15 , wherein said reaction mixture further comprises PCR reagents and flap reagents for amplifying and detecting a point mutation in said second genomic locus.
17. 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 said first primer:
i. comprises a 3′ terminal nucleotide that base pairs with a point mutation in said genomic locus; and
ii. comprises a nucleotide sequence that is fully complementary to a sequence in said locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide;
b) flap assay reagents comprising a flap endonuclease, a FRET cassette and a flap oligonucleotide that comprises a nucleotide that base pairs with said point mutation;
c) said nucleic acid sample, wherein said nucleic acid sample comprises both wild type copies of said genomic locus and mutant copies of said genomic locus that have a point mutation relative to said wild type copies of the genomic locus;
wherein said reaction mixture is characterized in that it can amplify and detect the presence of said mutant copies of said genomic locus in said sample.
18. The reaction mixture of claim 17 , wherein the reaction mixture optionally comprises, an invasive oligonucleotide distinct from said first primer that has a 3′ terminal nucleotide that base pairs with said point mutation.
19. A kit comprising:
a) PCR reagents that include a first primer and a second primer, wherein said first primer comprises a 3′ terminal nucleotide that base pairs with a point mutation in a genomic locus and also comprises a nucleotide sequence that is fully complementary to a sequence in said genomic locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide; and
b) flap assay reagents that include an invasive oligonucleotide having a 3′ terminal nucleotide that base pairs with said point mutation and a flap oligonucleotide that comprises a nucleotide that base pairs with said point mutation.
20. The kit of claim 19 , wherein said kit further comprises PCR and 5 flap reagents for amplification and detection of a control nucleic acid.
21. The kit of claim 20 , further comprising instructions for performing 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 said genomic locus that have a point mutation relative to said wild type copies of the genomic locus, to produce an amplified sample;
wherein:
i. said amplifying is done using a first primer and a second primer; and
ii. said first primer comprises a 3′ terminal nucleotide that base pairs with said point mutation and also comprises a nucleotide sequence that is fully complementary to a sequence in said locus with the exception of a single base mismatch within 6 bases of said 3′ terminal nucleotide;
b) detecting the presence of said product in said amplified sample using a flap assay that employs:
i. an invasive oligonucleotide having a 3′ terminal nucleotide that base pairs with said point mutation and
ii. a flap oligonucleotide that comprises a nucleotide that base pairs with said point mutation.