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 a first nucleic acid and a second nucleic acid, wherein the first nucleic acid and the second nucleic acid are derived from the same genomic locus and differ by one or more nucleotide substitutions;
wherein:
i. said amplifying is done using a first primer and a second primer, which are each complementary to a sequence in said locus; and
ii. said first primer comprises a 3′ terminal nucleotide that base pairs with a nucleotide substitution 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 amplification products that have said nucleotide substitution during amplification using a flap assay that employs:
i. said first primer; and
ii. a flap oligonucleotide that comprises a nucleotide that base pairs with said nucleotide substitution.
2. The method of claim 1 , wherein the nucleotide substitution is a single nucleotide polymorphism.
3. The method of claim 1 , wherein the nucleotide substitution is a mutation.
4. The method of claim 1 , wherein said sample contains at least 100 times more of the first nucleic acid than the second nucleic acid.
5. The method of claim 1 , further comprising normalizing the amount of the first or second nucleic acid relative to the amount of a control nucleic acid present in the sample.
6. The method of claim 5 , wherein said control nucleic acid is different from said genomic locus.
7. The method of claim 5 , wherein said control nucleic acid is detected by a second flap assay.
8. The method of claim 5 , wherein said genomic locus is associated with cancer.
9. The method of claim 5 , wherein said sample is obtained from a human.
10. The method of claim 9 , wherein sample is DNA extracted from stool.
11. The method of claim 5 , 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.
12. The method of claim 11 , wherein the mismatch is a G:T, G:G, A:G, T:G, G:A, T:T, T:C, A:C, C:T, A:A, C:A or C:C mismatch.
13. The method of claim 5 , 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.
14. The method of claim 13 , wherein said reaction mixture further comprises PCR reagents and flap reagents for amplifying and detecting a second genomic locus.
15. The method of claim 1 , further comprising making a diagnosis of colon cancer or adenoma based on results obtained from step (b).