Sequencing method employing ternary complex destabilization to identify cognate nucleotides
First Claim
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1. A method for identifying a nucleotide of a nucleic acid, comprising:
- (a) providing a solid support that is attached to a primed template nucleic acid molecule, wherein the solid support is in contact with a first reaction mixture that comprises a polymerase and a plurality of different nucleotide types,wherein the primed template nucleic acid molecule, the polymerase and one of the plurality of different nucleotide types form a stabilized ternary complex on the solid support;
(b) progressively removing nucleotide types of the plurality of different nucleotide types from contact with the primed template nucleic acid molecule on the solid support;
(c) monitoring stability of the ternary complex on the solid support during the progressive removal of the nucleotide types, without incorporating any nucleotide into the primer; and
(d) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from step (c).
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Abstract
Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.
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Citations
16 Claims
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1. A method for identifying a nucleotide of a nucleic acid, comprising:
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(a) providing a solid support that is attached to a primed template nucleic acid molecule, wherein the solid support is in contact with a first reaction mixture that comprises a polymerase and a plurality of different nucleotide types, wherein the primed template nucleic acid molecule, the polymerase and one of the plurality of different nucleotide types form a stabilized ternary complex on the solid support; (b) progressively removing nucleotide types of the plurality of different nucleotide types from contact with the primed template nucleic acid molecule on the solid support; (c) monitoring stability of the ternary complex on the solid support during the progressive removal of the nucleotide types, without incorporating any nucleotide into the primer; and (d) identifying the nucleotide that comprises the base complementary to the next base of the template strand using results from step (c). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification