Detection and quantification of biomolecules using mass spectrometry
First Claim
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1. An amplification 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 sequence with a set of oligonucleotide primers comprising a 3′
end and a 5′
end, wherein a first primer comprises a sequence complementary to a region in one strand of the target nucleic acid sequence, and a second primer comprises a sequence complementary to a region in a second strand of the target nucleic acid sequence;
(b) further contacting the sample with at least one detector oligonucleotide, comprising a 3′
end and a 5′
end, and comprising a sequence complementary to a region of the target nucleic acid between the region of the target sequence complementary to the sequence of the first primer and the region of the target sequence complementary to the sequence of the second primer, 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 the target nucleic acid sequence, thereby forming a mixture of duplexes under hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the primer oligonucleotides and to the detector oligonucleotide such that the 3′
end of each primer oligonucleotide is upstream of the 5′
end of the detector oligonucleotide annealed to the same nucleic acid strand;
(c) exposing the products of step (b) to a template-dependent polymerizing agent having 5′
to 3′
nuclease activity under conditions permissive for amplification, whereby the primer oligonucleotides are extended to produce extension products and the detector oligonucleotide is cleaved and releases a fragment comprising the nucleotide sequence that is non-complementary to the target nucleic acid sequence, thereby producing a mass-distinguishable product; and
(d) detecting the presence or absence of the mass-distinguishable by mass spectrometry, thereby detecting the presence or absence of the target 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 takes advantage of the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and releases labels for detection by mass spectrometry. This process is easily incorporated into a polymerase chain reaction (PCR) amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.
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Citations
18 Claims
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1. An amplification 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 sequence with a set of oligonucleotide primers comprising a 3′
end and a 5′
end, wherein a first primer comprises a sequence complementary to a region in one strand of the target nucleic acid sequence, and a second primer comprises a sequence complementary to a region in a second strand of the target nucleic acid sequence;(b) further contacting the sample with at least one detector oligonucleotide, comprising a 3′
end and a 5′
end, and comprising a sequence complementary to a region of the target nucleic acid between the region of the target sequence complementary to the sequence of the first primer and the region of the target sequence complementary to the sequence of the second primer, 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 the target nucleic acid sequence, thereby forming a mixture of duplexes under hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the primer oligonucleotides and to the detector oligonucleotide such that the 3′
end of each primer oligonucleotide is upstream of the 5′
end of the detector oligonucleotide annealed to the same nucleic acid strand;(c) exposing the products of step (b) to a template-dependent polymerizing agent having 5′
to 3′
nuclease activity under conditions permissive for amplification, whereby the primer oligonucleotides are extended to produce extension products and the detector oligonucleotide is cleaved and releases a fragment comprising the nucleotide sequence that is non-complementary to the target nucleic acid sequence, thereby producing a mass-distinguishable product; and(d) detecting the presence or absence of the mass-distinguishable by mass spectrometry, thereby detecting the presence or absence of the target sequence in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification
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Current AssigneeAgena Bioscience Inc. (Mesa Laboratories Inc.)
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Original AssigneeAgena Bioscience Inc. (Mesa Laboratories Inc.)
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InventorsVan Den Boom, Dirk Johannes, Oeth, Paul Andrew, Mahboubi, Payam
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Primary Examiner(s)Riley, Jezia
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Application NumberUS13/766,482Publication NumberTime in Patent Office846 DaysField of Search435/6.1, 435/91.1, 536/23.1, 536/24.3, 536/26.6US Class Current1/1CPC Class CodesC12Q 1/6823 Release of bound markersC12Q 1/6827 for detection of mutation o...C12Q 1/686 Polymerase chain reaction [...C12Q 1/6872 involving mass spectrometryC12Q 1/6883 for diseases caused by alte...C12Q 2521/319 ExonucleaseC12Q 2525/161 incorporating target specif...C12Q 2531/113 PCRC12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2545/114 involving a quantitation stepC12Q 2561/101 TaqmanC12Q 2565/627 being a mass spectrometer
