Patent ID: 8133701

Claim:
A method of detecting the presence or absence of a target nucleic acid sequence in a sample, comprising the steps of: (a) contacting a sample comprising a target nucleic acid with an (i) oligonucleotide primer, comprising a 3′ end and a 5′ end, and comprising a sequence complementary to a region of the target nucleic acid and a (ii) detector oligonucleotide, comprising a 3′ end and a 5′ end, and comprising a sequence complementary to a second region of the target nucleic acid sequence which comprises a non-cleavable nucleotide incorporated at its 5′ end and a contiguous nucleotide sequence that is non-complementary to the target nucleic acid sequence linked to the 5′ end of the sequence complementary to the second region of the target nucleic acid sequence, thereby forming a (iii) mixture of duplexes under hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the oligonucleotide primer and to the detector oligonucleotide such that the 3′ end of the oligonucleotide primer is upstream of the 5′ end of the detector oligonucleotide; (b) exposing the sample of step (a) to a cleavage agent under conditions sufficient to cleave and release from the annealed detector oligonucleotide, a fragment comprising the nucleotide sequence that is non-complementary to the target nucleic acid sequence thereby producing a mass-distinguishable product; and (c) detecting the presence or absence of the mass-distinguishable product by mass spectrometry, thereby detecting the presence or absence of the target nucleic acid sequence in the sample.