QUANTITATIVE NUCLEASE PROTECTION SEQUENCING (qNPS)
First Claim
1. A method of detecting at least one target in a biological sample comprising(i) contacting said sample with at least one nuclease protection probe (NPP) which specifically binds to said target,(ii) exposing said sample to one or more reagents under conditions that are effective to eliminate any unbound NPP,(iii) optionally separating the bound NPP from the target, and(iv) sequencing said NPP, a complement thereof, or a molecule incorporating said NPP or a compliment.
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Abstract
The present invention provides a new approach, quantitative Nuclease Protection Sequencing (qNPS™), for addressing several challenges that face sequencing and which provides improvements for research and diagnostic applications. The method uses a lysis-only nuclease protection assay to generate nucleic acid, e.g., DNA probes for sequencing, which can be coupled to gene-specific tags to permit the identification of the gene without necessitating the sequencing of the nuclease protection probe itself and/or can be coupled to experiment-specific tags whereby samples from different patients can be combined into a single run. The disclosed qNPS makes sequencing fixed or insoluble samples possible and affordable as a research and discovery tool and as a diagnostic test.
41 Citations
71 Claims
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1. A method of detecting at least one target in a biological sample comprising
(i) contacting said sample with at least one nuclease protection probe (NPP) which specifically binds to said target, (ii) exposing said sample to one or more reagents under conditions that are effective to eliminate any unbound NPP, (iii) optionally separating the bound NPP from the target, and (iv) sequencing said NPP, a complement thereof, or a molecule incorporating said NPP or a compliment.
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24. A method for detecting at least one nucleic acid target in a biological sample comprising
(i) contacting said sample with at least one nuclease protection probe (NPP) which is a nucleic acid molecule that specifically hybridizes to said nucleic acid target under conditions sufficient to facilitate binding of said target to said NPP, (ii) exposing said sample to one or more nucleases under conditions that are effective to eliminate any unbound NPP, (iii) optionally separating the bound NPP from the target (v) amplifying said NPP or adduct containing said NPP and (v) sequencing said NPP.
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39. A method of detecting at least one target in a biological sample comprising
(i) contacting said sample with at least one nuclease protection probe (NPP) which specifically binds to said target, (ii) exposing said sample to one or more reagents under conditions that are effective to eliminate any unbound NPP and target that is not hybridized to the NPP, (iii) optionally separating the bound NPP from the target, (iv) optionally amplifying said NPP, or a complement to the NPP, or the target, or an adduct containing the NPP or target or complement to the NPP, and (v) sequencing said NPP, or the target, or a complement to the NPP or an adduct containing the NPP or the target, or a complement to the NPP.
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45. A sequencible adduct comprising
a nuclease protection probe (NPP) comprising a polynucleotide sequence which hybridizes to a biological target; -
a first tag comprising a polynucleotide sequence which extends from the 3′
end of said NPP via the 5′
end of the tag sequence; and
optionallya second tag comprising a polynucleotide sequence which extends from the 3′
end of said first tag. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)
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70. A method of detecting at least one target in a biological sample comprising
(i) contacting said sample with at least one linear nuclease protection probe (NPP), the ends of which specifically binds to said target such that the 5′ - and 3′
end are hybridized to adjacent bases of the target,(ii) ligating said NPP to form a circular oligonucleotide, (iii) optionally dissociating the circular NPP, hybridizing a second molecule of linear NPP to the target, and ligating, (iv) optionally repeating (iv) in successive cycles, (v) adding a nuclease to destroy all linear single stranded oligonucleotide in the sample, and (vi) cleaving the circular NPP to linearize said NPP, and (vii) sequencing the linear NPP.
- and 3′
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71. A method of detecting at least one target in a biological sample comprising
(i) contacting said sample with at least one nuclease protection probe (NPP) which specifically binds to said target, (ii) exposing said sample to one or more reagents under conditions that are effective to eliminate any unbound NPP and target that is not hybridized to the NPP, (iii) optionally separating the bound NPP from the target, (iv) optionally amplifying said NPP, or a complement to the NPP, or the target, or an adduct comprising a nuclease protection probe (NPP) comprising a polynucleotide sequence which hybridizes to a biological target; -
a first tag comprising a polynucleotide sequence which extends from the 3′
end of said NPP via the 5′
end of the tag sequence; and
optionallya second tag comprising a polynucleotide sequence which extends from the 3′
end of said first tag, and(v) sequencing said NPP, or the target, or a complement to the NPP or said adduct.
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Specification