Nucleic acid sequencing methods and systems
First Claim
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1. A method of sequencing at least a portion of a template nucleic acid, the method comprising:
- (a) providing a sequencing reaction mixture comprising;
(i) the template nucleic acid;
(ii) a complementary nucleic acid that is at least partially complementary to the template nucleic acid, wherein the complementary nucleic acid is hybridized to the template nucleic acid;
(iii) an exonuclease that comprises template dependent nucleobase binding activity; and
,(iv) at least one labeled nucleobase;
(b) permitting the sequencing reaction mixture to react, such that the exonuclease transiently binds the labeled nucleobase in a template dependent manner, wherein the exonuclease moves along the template as it digests the complementary nucleic acid, wherein said nucleobase is not incorporated by said exonuclease;
(c) detecting transient binding of the labeled nucleobase by the exonuclease; and
, (d) determining a sequence of the template, or a portion thereof, based upon said detecting.
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Abstract
Sequencing methods that use an exonuclease that comprises template dependent nucleobase binding activity are provided. Related compositions and sequencing systems are also provided.
72 Citations
17 Claims
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1. A method of sequencing at least a portion of a template nucleic acid, the method comprising:
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(a) providing a sequencing reaction mixture comprising; (i) the template nucleic acid; (ii) a complementary nucleic acid that is at least partially complementary to the template nucleic acid, wherein the complementary nucleic acid is hybridized to the template nucleic acid; (iii) an exonuclease that comprises template dependent nucleobase binding activity; and
,(iv) at least one labeled nucleobase; (b) permitting the sequencing reaction mixture to react, such that the exonuclease transiently binds the labeled nucleobase in a template dependent manner, wherein the exonuclease moves along the template as it digests the complementary nucleic acid, wherein said nucleobase is not incorporated by said exonuclease;
(c) detecting transient binding of the labeled nucleobase by the exonuclease; and
, (d) determining a sequence of the template, or a portion thereof, based upon said detecting. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification