Methods of screening nucleic acids using mass spectrometry
First Claim
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1. A method of detecting mutations in a target nucleic acid comprising:
- obtaining a target nucleic acid in single-stranded form;
hybridizing the single-stranded target nucleic acid to one or more sets of fragmenting probes to form hybrid target nucleic acid/fragmenting probe complexes comprising at least one double-stranded region and at least one single-stranded region;
nonrandomly fragmenting the target nucleic acid by cleaving the hybrid target nucleic acid/fragmenting probe complexes at every single-stranded region with at least one single-strand-specific cleaving reagent to form a set of nonrandom length fragments (NLFs); and
determining masses of the members of the set using mass spectrometry.
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Abstract
Methods for screening nucleic acids for mutations by analyzing nonrandomly fragmented nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods of detecting mutations. Kits for performing the methods are provided.
205 Citations
43 Claims
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1. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
hybridizing the single-stranded target nucleic acid to one or more sets of fragmenting probes to form hybrid target nucleic acid/fragmenting probe complexes comprising at least one double-stranded region and at least one single-stranded region;
nonrandomly fragmenting the target nucleic acid by cleaving the hybrid target nucleic acid/fragmenting probe complexes at every single-stranded region with at least one single-strand-specific cleaving reagent to form a set of nonrandom length fragments (NLFs); and
determining masses of the members of the set using mass spectrometry. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
nonrandomly fragmenting the target nucleic acid with one or more restriction endonucleases to form a set of nonrandom length fragments (NLFs);
hybridizing one or more of the set of NLFs or a subset thereof to one or more oligonucleotide probes, wherein each of the oligonucleotide probes comprises a nucleic acid comprising a single-stranded region and a first binding moiety, binding the first binding moiety to a second binding moiety attached to a solid support either before or after the hybridizing step;
isolating the set or subset of single-stranded NLFs; and
determining masses of the members of the set using mass spectrometry. - View Dependent Claims (13, 14, 15, 16)
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17. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
hybridizing the single-stranded target nucleic acid to one or more restriction site probes to form hybridized target nucleic acids having double-stranded regions, wherein the restriction site probes have hybridized to the single-stranded target nucleic acid and at least one single-stranded region;
nonrandomly fragmenting the hybridized target nucleic acids by contacting it with one or more restriction endonucleases that cleave at restriction sites within the double-stranded regions to produce a set of single-straneded nonrandom length fragments (NLFs); and
determining masses of the members of the set using mass spectrometry. - View Dependent Claims (18, 19)
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20. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
exposing the target nucleic acid to conditions permitting folding of the single-stranded target nucleic acid to form a three-dimensional structure having intramolecular secondary and tertiary interactions;
nonrandomly fragmenting the folded target nucleic acid with at least one structure-specific endonuclease to form a set of single-stranded nonrandom length fragments (NLFs);
modifying either the target nucleic acid or the set of single-stranded NLFs such that members of the set of single-stranded NLFs comprise a single-stranded nucleotide sequence and at least one first binding moiety;
binding the first binding moiety to a second binding moiety attached to a solid support;
isolating the set of single-stranded NLFs; and
determining masses of the members of the set using mass spectrometry. - View Dependent Claims (21)
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22. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
exposing the nucleic acid to conditions permitting folding of the single-stranded target nucleic acid to form a three-dimensional structure having intramolecular secondary and tertiary interactions;
nonrandomly fragmenting the folded target nucleic acid with at least one structure-specific endonuclease to form a set of single-stranded nonrandom length fragments (NLFs);
hybridizing one or more of the set of NLFs to one or more capture probes, wherein the capture probes comprise a single-stranded nucleotide sequence and a first binding moiety;
binding the first binding moiety to a second binding moiety attached to a solid support either before or after the hybridizing step;
isolating a set of single-stranded NLFs; and
determining masses of the members of the set using mass spectrometry. - View Dependent Claims (23, 24)
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25. A method of detecting mutations in a target nucleic acid comprising:
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obtaining a target nucleic acid in single-stranded form;
hybridizing the single-stranded target nucleic acid to one or more wild type probes;
nonrandomly fragmenting the target nucleic acid with one or more mutation-specific cleaving reagents that specifically cleave at any regions of nucleotide mismatch that form between the target nucleic acid and any of the wild type probes to form a set of single-stranded nonrandom length fragments (NLFs); and
determining masses of the members of the set using mass spectrometry - View Dependent Claims (26, 27, 28, 29)
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30. A method of detecting mutations in a target nucleic acid comprising:
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nonrandomly fragmenting the target nucleic acid with one or more restriction endonucleases to form a set of double-stranded nonrandom length fragments (NLFs), wherein the nonrandomly fragmenting further comprises including volatile salts in the restriction buffer; and
determining masses of the members of the set of double-stranded NLFs, wherein the determining does not involve sequencing of the target nucleic acid.
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31. A method of detecting mutations in a double-stranded target nucleic acid comprising:
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nonrandomly fragmenting the target nucleic acid using one or more restriction endonucleases to form a first set of nonrandom length fragments (NLFs);
hybridizing members of the first set of NLFs to a set of wild type probes;
nonrandomly fragmenting one or more members of the set of NLFs with one or more mutation-specific cleaving reagents that specifically cleave at any regions of nucleotide mismatch that form between members of the first set of NLFs and complementary members of the set of wild type probes, wherein the nonrandomly fragmenting step forms a second set of NLFs; and
determining masses of members of the second set of NLFs using mass spectrometry, wherein the determining does not require sequencing of the target nucleic acid. - View Dependent Claims (32, 33, 34, 35)
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36. A method of detecting mutations in a target nucleic acid comprising:
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nonrandomly fragmenting the target nucleic acid, using a solution comprising one or more volatile salts to form a set of nonrandom length fragments (NLFs); and
determining masses of members of the set of NLFs using mass spectrometry, wherein the determining does not involve sequencing of the target nucleic acid.
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37. A kit for detecting mutations in one or more target nucleic acids in a sample comprising:
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(a) one or more sets of fragmenting probes, wherein the fragmenting probes are complementary to a sequence of one or more of the target nucleic acids;
(b) a single-strand specific cleaving regent;
(c) a solid support for isolating single-stranded target nucleic acids that have been nonrandomly fragmented into single-stranded nonrandom length fragments; and
(d) matrix for performing mass spectrometry analyses. - View Dependent Claims (38, 39)
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40. A kit for detecting mutations in one or more target nucleic acids in a sample comprising:
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(a) one or more sets of restriction site probes, wherein the probes comprise a single-stranded sequence capable of hybridizing to a sequence of the one or more target nucleic acids;
(b) one or more restriction endonucleases that cleave at restriction sites within the restriction site probes; and
(c) a solid support for isolating single-stranded target nucleic acids that have been nonrandomly fragmented into single-stranded nonrandom length fragments. - View Dependent Claims (41, 42, 43)
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Specification