Method and system for sequencing nucleic acids
First Claim
1. A method of determining the identity of the next correct nucleotide for a primed template nucleic acid molecule, the method comprising:
- (a) providing a template nucleic acid molecule primed with a primer;
(b) serially contacting the primed template nucleic acid molecule with a plurality of reaction mixtures, each of the reaction mixtures comprising a polymerase and a different combination of two and only two or three and only three different test nucleotides, under conditions that stabilize ternary complexes comprising the primed template nucleic acid molecule, the polymerase and a next correct nucleotide, while precluding incorporation of any nucleotide into the primer;
(c) detecting interaction of the polymerase with the primed template nucleic acid molecule without chemical incorporation of any nucleotide into the primer of the primed template nucleic acid molecule, to determine whether ternary complexes form; and
(d) determining whether a test nucleotide that is common to at least two of the reaction mixtures is the next correct nucleotide for the primed template nucleic acid molecule using the detected interaction.
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Accused Products
Abstract
Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents.
103 Citations
20 Claims
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1. A method of determining the identity of the next correct nucleotide for a primed template nucleic acid molecule, the method comprising:
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(a) providing a template nucleic acid molecule primed with a primer; (b) serially contacting the primed template nucleic acid molecule with a plurality of reaction mixtures, each of the reaction mixtures comprising a polymerase and a different combination of two and only two or three and only three different test nucleotides, under conditions that stabilize ternary complexes comprising the primed template nucleic acid molecule, the polymerase and a next correct nucleotide, while precluding incorporation of any nucleotide into the primer; (c) detecting interaction of the polymerase with the primed template nucleic acid molecule without chemical incorporation of any nucleotide into the primer of the primed template nucleic acid molecule, to determine whether ternary complexes form; and (d) determining whether a test nucleotide that is common to at least two of the reaction mixtures is the next correct nucleotide for the primed template nucleic acid molecule using the detected interaction. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of sequencing a primed template nucleic acid, comprising:
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(a) contacting a primed template nucleic acid with a polymerase and a first combination of two and only two or three and only three types of test nucleotides under conditions that form a stabilized ternary complex between the polymerase, primed template nucleic acid and a test nucleotide that is complementary to the next base of the primed template nucleic acid; (b) detecting the ternary complex while precluding incorporation of test nucleotides into the primer; (c) repeating steps (a) and (b) using the primed template nucleic acid, a polymerase and a second combination of two or three types of test nucleotides, wherein the second combination is different from the first combination; (d) incorporating into the primer, after step (c), a nucleotide that is complementary to the next base; and (e) repeating steps (a) through (d) to identify a sequence of the primed template nucleic acid. - View Dependent Claims (17, 18, 19, 20)
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Specification