Patent ID: 6110709
Filing Date: 2000-08-29
Classification: C12Q

Abstract:
A method for detecting the presence or absence of a single nucleotide polymorphism in a nucleic acid molecule, said method comprising the steps of:(a) providing a nucleic acid molecule comprising a first strand, a second strand, and a nucleotide position suspected of comprising a polymorphism;(b) amplifying a portion of said nucleic acid molecule by PCR using a first primer and a second primer, wherein (i) said first primer comprises a 3' region that is complementary to a first region of said first strand, the 5' end of said first region being positioned within 15 nucleotides 3' of said nucleotide position, and said 3' region of said first primer comprising at least one nucleotide mismatch with said first region within 15 nucleotides of said nucleotide position, (ii) said second primer comprises a 3' region that is complementary to a second region of said second strand, the 5' end of said second region being positioned 3' of said nucleotide position, and (iii) said amplification generates a PCR product comprising at least one point mutation, relative to the sequence of said nucleic acid molecule, that forms a portion of a restriction endonuclease recognition site in the presence of a single polymorphic nucleotide in said nucleotide position;(c) labeling one end of one strand of the product of step (b) with a detectable label to generate a labeled PCR product comprising a labeled strand;(d) treating said labeled PCR product with a restriction endonuclease that recognizes said restriction endonuclease recognition site to generate a digestion product;(e) denaturing and contacting said digestion product with a probe that (i) is complementary to a first segment of said labeled strand that is on the opposite side of said nucleotide position from said detectable label, within said labeled strand, (ii) is not complementary to said labeled strand between said nucleotide position and said detectable label, and (iii) is immobilized to a binding element on a solid support; and(f) assaying for said detectable label bound to said binding element, wherein the absence of said label bound to said binding element indicates the presence of said single nucleotide polymorphism in said nucleic acid molecule, and the presence of said label bound to said binding element indicates the absence of said single nucleotide polymorphism in said nucleic acid molecule.