DNA diagnostics based on mass spectrometry
First Claim
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1. A process for identifying the presence or absence of a mutation in a target sequence in a nucleic acid molecule, comprising the steps of:
- a) hybridizing a nucleic acid molecule comprising the target nucleic acid sequence with an oligonucleotide probe, which is complementary to a region of the target nucleic acid sequence that can contain a mutation, thereby forming a heteroduplex;
b) contacting the heteroduplex with a single strand specific endonuclease, which can cleave the heteroduplex at a site of a mismatch, if present, to produce a cleavage product comprising a cleaved probe, a cleaved target nucleic acid sequence, or both; and
c) detecting the product of step b) by mass spectrometry, thereby identifying the presence or absence of a mutation in the target nucleic acid sequence.
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Abstract
Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and sequences in the molecules are provided. Depending upon the sequence to be detected, the processes, for example, can be used to diagnose a genetic disease or a chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogen, or for determining identity or heredity.
289 Citations
45 Claims
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1. A process for identifying the presence or absence of a mutation in a target sequence in a nucleic acid molecule, comprising the steps of:
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a) hybridizing a nucleic acid molecule comprising the target nucleic acid sequence with an oligonucleotide probe, which is complementary to a region of the target nucleic acid sequence that can contain a mutation, thereby forming a heteroduplex;
b) contacting the heteroduplex with a single strand specific endonuclease, which can cleave the heteroduplex at a site of a mismatch, if present, to produce a cleavage product comprising a cleaved probe, a cleaved target nucleic acid sequence, or both; and
c) detecting the product of step b) by mass spectrometry, thereby identifying the presence or absence of a mutation in the target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A process for identifying the presence or absence of one or more mutations in a plurality of target nucleic acid sequences, comprising the steps of:
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a) hybridizing a plurality of nucleic acid molecules comprising the target nucleic acid sequences with a plurality of oligonucleotide probes, which are complementary to regions of the target nucleic acid sequences that can contain a mutation, wherein a probe that hybridizes to one target nucleic acid sequence in the plurality is distinguishable from each probe that hybridizes to a different target nucleic acid sequence, thereby forming one or more heteroduplexes, which are distinguishable;
b) contacting the one or more heteroduplexes with a single strand specific endonuclease, which can cleave a heteroduplex at a site of a mismatch, if present, to produce a cleavage product comprising a cleaved probe, a cleaved target nucleic acid, or both; and
c) detecting the products of step b) by mass spectrometry, thereby identifying the presence or absence of a mutation in each target nucleic acid sequence in the plurality. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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44. A method of detecting mutations in a target nucleic acid molecule comprising:
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hybridizing the target nucleic acid molecule to one or more probes;
fragmenting the target nucleic acid with a reagent that specifically cleaves at any regions of nucleotide mismatch that form between molecule target nucleic acid molecule and any of the probes to produce a set of fragments; and
determining masses of the members of said set by mass spectrometry, thereby identifying any mutations in the target nucleic acid molecule. - View Dependent Claims (45)
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Specification