Sequencing method for rapid identification and processing of cognate nucleotide pairs
First Claim
1. A method of determining the identity of first and second correct nucleotides respectively comprising bases complementary to the next two bases of a template strand immediately downstream of a primer of a primed template nucleic acid molecule, said method comprising the steps of:
- (a) conducting a plurality of cycles of contacting a first primed template nucleic acid molecule with a reaction mixture comprising a polymerase and, for each cycle, a different test nucleotide, and removing any polymerase and nucleotide that may have bound to the first primed template nucleic acid molecule,wherein the polymerase does not incorporate any of the different test nucleotides into the primer of the first primed template nucleic acid molecule during any of the plurality of cycles;
(b) measuring, for the cycles in step (a), signals indicating the magnitudes of formation of stabilized ternary complexes comprising binding of the first primed template nucleic acid molecule to the polymerase and the different test nucleotides; and
(c) determining the identities of the first correct nucleotide downstream of the primer, and the second correct nucleotide downstream of the primer using the measured signals from step (b), wherein the test nucleotide associated with the highest magnitude of stabilized ternary complex formation is identified as the first correct nucleotide downstream of the primer, and the test nucleotide associated with the second-highest magnitude of stabilized ternary complex formation is identified as the second correct nucleotide downstream of the primer.
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Abstract
Provided are methods and systems for reducing the time needed for sequencing nucleic acids. The approach relies on detecting formation of nucleotide-specific ternary complexes comprising a polymerase (e.g., a DNA polymerizing enzyme), a primed template nucleic acid molecule, 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, as well as the subsequent next correct nucleotide from a cycle of examining four different nucleotides without requiring chemical incorporation of any nucleotide into the primer.
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Citations
27 Claims
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1. A method of determining the identity of first and second correct nucleotides respectively comprising bases complementary to the next two bases of a template strand immediately downstream of a primer of a primed template nucleic acid molecule, said method comprising the steps of:
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(a) conducting a plurality of cycles of contacting a first primed template nucleic acid molecule with a reaction mixture comprising a polymerase and, for each cycle, a different test nucleotide, and removing any polymerase and nucleotide that may have bound to the first primed template nucleic acid molecule, wherein the polymerase does not incorporate any of the different test nucleotides into the primer of the first primed template nucleic acid molecule during any of the plurality of cycles; (b) measuring, for the cycles in step (a), signals indicating the magnitudes of formation of stabilized ternary complexes comprising binding of the first primed template nucleic acid molecule to the polymerase and the different test nucleotides; and (c) determining the identities of the first correct nucleotide downstream of the primer, and the second correct nucleotide downstream of the primer using the measured signals from step (b), wherein the test nucleotide associated with the highest magnitude of stabilized ternary complex formation is identified as the first correct nucleotide downstream of the primer, and the test nucleotide associated with the second-highest magnitude of stabilized ternary complex formation is identified as the second correct nucleotide downstream of the primer. - 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)
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26. A method of determining the identity of first and second correct nucleotides respectively comprising bases complementary to the next two bases of a template strand immediately downstream of a primer in a primed template nucleic acid molecule, said method comprising the steps of:
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(a) conducting a plurality of cycles of contacting a first primed template nucleic acid molecule with a reaction mixture comprising a polymerase and, for each cycle, a different pair of test nucleotides, and removing any polymerase and nucleotide that may have bound to the first primed template nucleic acid molecule, wherein the polymerase does not incorporate any of the different test nucleotides into the primer of the first primed template nucleic acid molecule during any of the four cycles; (b) measuring, for the cycles in step (a), signals indicating the magnitudes of formation of stabilized ternary complexes comprising binding of the first primed template nucleic acid molecule to the polymerase and the test nucleotides among the different pairs of test nucleotides; and (c) determining the identities of the first correct nucleotide downstream of the primer, and the second correct nucleotide downstream of the primer using the measured signals from step (b), wherein the test nucleotide associated with the highest magnitude of stabilized ternary complex formation is identified as the first correct nucleotide downstream of the primer, and the test nucleotide associated with the second-highest magnitude of stabilized ternary complex formation is identified as the second correct nucleotide downstream of the primer. - View Dependent Claims (27)
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Specification