Methods and kits for analyzing polynucleotide sequences
First Claim
1. A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:
- a) exposing a duplex comprising a template and a primer to a polymerase and one or more nucleotides comprising a detectable label under conditions sufficient for template-dependent nucleotide addition to said primer, the primer being hybridized to a first region of the template, wherein said duplex is individually optically resolvable;
b) identifying nucleotide incorporated into said primer;
c) repeating steps a) and b), thereby determining a nucleotide sequence;
d) removing the primer from the template;
e) exposing the template to a second primer capable of hybridizing to the first region of the template to form a template/primer duplex, and repeating steps a) through c) to resequence a portion of the template, thereby increasing the accuracy of nucleic acid sequencing.
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Abstract
The present invention features methods for analyzing a sequence of a target polynucleotide by detecting incorporation of a nucleotide into its complementary strand, where the polynucleotides may be bound at high density and at single molecule resolution. The invention also features labeling moieties and blocking moieties, which facilitate chain termination or choking. Certain aspects provide for temporal detection of the incorporations; some allow for asynchronous analysis of a plurality of target polynucleotides and the use of short sequencing cycles. Surface chemistry aspects of the sequencing methods are also provided. The method may also be used in kits, said kits designed to carry out and facilitate the methods provided herein.
504 Citations
22 Claims
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1. A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:
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a) exposing a duplex comprising a template and a primer to a polymerase and one or more nucleotides comprising a detectable label under conditions sufficient for template-dependent nucleotide addition to said primer, the primer being hybridized to a first region of the template, wherein said duplex is individually optically resolvable; b) identifying nucleotide incorporated into said primer; c) repeating steps a) and b), thereby determining a nucleotide sequence; d) removing the primer from the template; e) exposing the template to a second primer capable of hybridizing to the first region of the template to form a template/primer duplex, and repeating steps a) through c) to resequence a portion of the template, thereby increasing the accuracy of nucleic acid sequencing. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of increasing accuracy of nucleic acid sequencing, the method comprising the steps of:
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a) exposing a duplex comprising a template and a plurality of primers to a polymerase and one or more nucleotide comprising a detectable label under conditions sufficient for template-dependent nucleotide addition to at least one of said plurality of primers, the plurality of primers being hybridized to a plurality of regions of the template, wherein said duplex is individually optically resolvable; b) identifying incorporated nucleotides; c) repeating steps a) and b), thereby determining a nucleotide sequence of at least one of said plurality of regions of the template; d) removing at least one of said plurality of primers from the template; e) exposing the template to a second plurality of primers capable of hybridizing to a first region of the template to form a template/primer duplex, and repeating steps a) and c) to resequence the at least one of said plurality of regions of the template, thereby increasing the accuracy of nucleic acid sequencing. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification