Detection and quantification of biomolecules using mass spectrometry
First Claim
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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 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.
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Abstract
The present invention is directed in part to a method for detecting a target nucleic acid using detector oligonucleotides detectable by mass spectrometry. This method uses the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and release labels for detection by mass spectrometry. This process is easily incorporated into a PCR amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.
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19 Claims
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1. A method of detecting the presence or absence of a target nucleic acid sequence in a sample, comprising the steps of:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification