Nucleic acid sequencing methods and systems
First Claim
1. A method of identifying a base in a primed template nucleic acid, comprising(a) contacting a primed template nucleic acid with a polymerase that is attached to a label moiety and a test nucleotide, under conditions that:
- (i) stabilize ternary complex formed between the polymerase, primed template nucleic acid and test nucleotide when the test nucleotide is complementary to the next base of the primed template nucleic acid while precluding incorporation of the test nucleotide into the primer, and(ii) destabilize binary complex formed between the primed template nucleic acid and the polymerase when the test nucleotide is not complementary to the next base of the primed template nucleic acid;
(b) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer, wherein the detecting comprises detecting the label moiety that is attached to the polymerase; and
(c) identifying the next base of the primed template nucleic acid from the detected ternary complex.
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Abstract
The present disclosure provides compositions, methods and systems for sequencing a template nucleic acid using a polymerase based, nucleic acid binding reaction involving examination of the interaction between a polymerase and template nucleic acid in the presence of one or more unlabeled nucleotides. The methods rely, in part, on identifying a base of a template nucleic acid during nucleic acid synthesis by controlling the sequencing reaction conditions. Template nucleic acid bases may be identified during an examination step followed by an optional incorporation step.
60 Citations
69 Claims
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1. A method of identifying a base in a primed template nucleic acid, comprising
(a) contacting a primed template nucleic acid with a polymerase that is attached to a label moiety and a test nucleotide, under conditions that: -
(i) stabilize ternary complex formed between the polymerase, primed template nucleic acid and test nucleotide when the test nucleotide is complementary to the next base of the primed template nucleic acid while precluding incorporation of the test nucleotide into the primer, and (ii) destabilize binary complex formed between the primed template nucleic acid and the polymerase when the test nucleotide is not complementary to the next base of the primed template nucleic acid; (b) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer, wherein the detecting comprises detecting the label moiety that is attached to the polymerase; and (c) identifying the next base of the primed template nucleic acid from the detected ternary complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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13. A method of identifying a base in a primed template nucleic acid, comprising
(a) contacting a primed template nucleic acid with a polymerase and a test nucleotide, under conditions that: -
(i) stabilize ternary complex formed between the polymerase, primed template nucleic acid and test nucleotide when the test nucleotide is complementary to the next base of the primed template nucleic acid while precluding incorporation of the test nucleotide into the primer, and (ii) destabilize binary complex between the primed template nucleic acid and the polymerase when the test nucleotide is not complementary to the next base of the primed template nucleic acid; (b) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer, wherein the detecting is carried out in the absence of label moieties on the test nucleotide and on the polymerase; and (c) identifying the next base of the primed template nucleic acid from the detected ternary complex. - View Dependent Claims (27, 28, 29, 30, 44, 45, 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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Specification