Patent ID: 7455968

Claim:
A method of determining whether a nucleotide at a target position in a single-stranded sample DNA sequence is complementary to the nucleotide on the 3′ end of an oligonucleotide probe, which comprises: (i) providing the oligonucleotide probe, wherein the nucleotide on the 3′ end of said oligonucleotide probe is a terminally blocked nucleotide such that 5′-3′ extension of said oligonucleotide probe cannot take place in the presence of said blocked nucleotide, and wherein said oligonucleotide probe is capable of hybridizing to said single-stranded sample DNA sequence; (ii) incubating said single-stranded sample DNA sequence with said oligonucleotide probe in the presence of (a) a DNA polymerase having 3′-5′ exonuclease activity and (b) nucleotide triphosphates, under conditions that allow hybridization of the oligonucleotide probe to the single-stranded sample DNA sequence, without PCR amplification, such that, when said blocked nucleotide of said oligonucleotide probe is complementary to the nucleotide at said target position of said single-stranded sample DNA sequence, said blocked nucleotide of said oligonucleotide probe and the nucleotide at said target position of said single-stranded sample DNA sequence form a base pair and said blocked nucleotide of said oligonucleotide probe is not cleaved from said oligonucleotide probe by the 3′-5′ exonuclease activity of said DNA polymerase; and when said blocked nucleotide of said oligonucleotide probe is not complementary to the nucleotide at said target position of said single-stranded sample DNA sequence, said blocked nucleotide of said oligonucleotide probe is cleaved from said oligonucleotide probe by the 3′-5′ exonclease activity of the said DNA polymerase, thereby allowing the 5′-3′ extension of said oligonucleotide probe and producing pyrophosphate; and (iii) detecting the cleavage of said blocked nucleotide of said oligonucleotide probe by detecting said pyrophosphate wherein the presence of said cleavage indicates that said nucleotide at said target position of a said single-stranded sample DNA sequence is not complementary to the nucleotide on the 3′ end of said oligonucleotide probe.