Solution-based methods and materials for sequence analysis by hybridization
First Claim
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1. A method of detecting a sequence of a target nucleic acid, comprising:
- (a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe;
target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support;
(b) detecting probe molecules that hybridize with the target nucleic acid, using a reader capable of detecting a single probe molecule that hybridizes to target nucleic acid; and
(c) detecting a sequence of the target nucleic acid by overlapping sequences of the information regions of at least two of the probe molecules contacted with the target in step (a).
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Abstract
Novel solution-based methods and materials, including apparatus, for sequence analysis by hybridization are provided.
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Citations
37 Claims
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1. A method of detecting a sequence of a target nucleic acid, comprising:
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(a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe;
target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support;
(b) detecting probe molecules that hybridize with the target nucleic acid, using a reader capable of detecting a single probe molecule that hybridizes to target nucleic acid; and
(c) detecting a sequence of the target nucleic acid by overlapping sequences of the information regions of at least two of the probe molecules contacted with the target in step (a). - 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, 26, 27, 28, 29, 30, 34, 35, 36, 37)
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31. A method of sequencing a target nucleic acid, comprising:
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(a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe;
target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support;
(b) covalently joining probe molecules that form contiguous probe;
target hybrids that are perfectly complementary to the target in the information region of the probe molecules; and
(c) detecting covalently joined probe molecules, using a reader capable of detecting the hybridization of a single probe molecule to target nucleic acid. - View Dependent Claims (32, 33)
(d) detecting a sequence of the target nucleic acid by overlapping at least two sequences generated by combining sequences of the information region of two probe molecules contacted with target nucleic acid in step (a). -
33. The method of claim 31 wherein before detection step (c), covalently joined probe molecules are separated from probe molecules that have not been covalently joined.
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Specification