Consecutive base single molecule sequencing
First Claim
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1. A method for single molecule nucleic acid sequencing, the method comprising:
- covalently bonding to a surface individually optically resolvable duplexes comprising a nucleic acid template and a primer hybridized thereto;
conducting a template-dependent sequencing reaction mediated by a polymerase to extend primers of plural said optically resolvable duplexes by at least three consecutive optically labeled nucleotides; and
detecting optically, by observation at known positions on said surface, the addition of labeled nucleotides to individual said duplexes thereby to determine the sequence of at least three bases of respective said templates with an accuracy of at least 70% with respect to a reference sequence.
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Abstract
The invention provides methods for sequencing polynucleotide molecules using single molecule sequencing techniques, where a plurality of labeled nucleotides are incorporated consecutively into an individual primer molecule.
118 Citations
46 Claims
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1. A method for single molecule nucleic acid sequencing, the method comprising:
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covalently bonding to a surface individually optically resolvable duplexes comprising a nucleic acid template and a primer hybridized thereto;
conducting a template-dependent sequencing reaction mediated by a polymerase to extend primers of plural said optically resolvable duplexes by at least three consecutive optically labeled nucleotides; and
detecting optically, by observation at known positions on said surface, the addition of labeled nucleotides to individual said duplexes thereby to determine the sequence of at least three bases of respective said templates with an accuracy of at least 70% with respect to a reference sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of sequencing a nucleic acid template comprising:
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(a) exposing a nucleic acid template hybridized to a primer having a 3′
end to (i) a polymerase which catalyzes nucleotide additions to the primer, and (ii) a labeled nucleotide under conditions to permit the polymerase to add the labeled nucleotide to the primer;
(b) detecting optically, by observation at known positions on said surface the labeled nucleotide added to the primer in step (a);
(c) removing the label from the labeled nucleotide;
(d) repeating steps (a), (b) and (c) thereby to determine the sequence of at least three bases of respective said templates with an accuracy of at least 70% with respect to a reference sequence. - View Dependent Claims (21, 22, 23)
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24. A method for single molecule nucleic acid sequencing, the method comprising:
- conducting a template-dependent sequencing reaction in which multiple labeled nucleotides are incorporated consecutively into a primer portion of a substrate-bound duplex thereby producing a sequence, the substrate-bound duplex comprising a nucleic acid template and primer hybridized thereto, wherein said duplex is individually optically resolvable on said substrate, and wherein the accuracy of the resulting sequence is at least 70% with respect to a reference sequence.
- View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
Specification