| 主权项 |
1. 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
(i) a pair of oligonucleotide primers, wherein a first oligonucleotide primer comprises a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a first extension product, and wherein a second oligonucleotide primer comprises a sequence complementary to a region in said first extension product and primes the synthesis of a nucleic acid strand complementary to said first extension product, and(ii) an oligonucleotide probe comprising at least two distinct portions,
a first portion comprised of standard nucleotides with or without nucleotide analogs that comprises a sequence that is at least partially complementary to a region of the target nucleic acid sequence wherein said first portion anneals within the target nucleic acid sequence bounded by said pair of oligonucleotide primers, and wherein said first portion is blocked at the 3′ terminus to prohibit extension by a nucleic acid polymerase and to prevent cleavage by a 3′ to 5′ exonuclease; anda second portion attached to the 5′ end of the first portion comprised of either nucleotides or non-nucleotides or both nucleotides and non-nucleotides, and comprises a sequence that is non-complementary to the target nucleic acid sequence, wherein said second portion also comprises an exonuclease-resistant modification; (b) amplifying said target nucleic acid sequence with a nucleic acid polymerase having 5′ to 3′ nuclease activity under conditions that allows annealing of said pair of oligonucleotide primers and said oligonucleotide probe to the target nucleic acid sequence and synthesis of primer extension products from said pair of oligonucleotide primers while the 5′ to 3′ nuclease activity of said nucleic acid polymerase is able to cleave and release from the annealed oligonucleotide probe, fragments containing the second portion of the oligonucleotide probe with or without additional nucleotides from the first portion of the oligonucleotide probe; (c) treating said fragments containing the second portion of the oligonucleotide probe with a 3′ to 5′ exonuclease that cleaves said fragments up to the exonuclease-resistant modification thereby producing a fragment having a unique mass-distinguishable size; and (d) detecting the presence or absence of the single fragment by mass spectrometry, thereby detecting the presence or absence of the target nucleic acid sequence in the sample. |
