Methods for Increasing Accuracy of Nucleic Acid Sequencing
First Claim
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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 nucleotide 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 invention provides methods for improving the fidelity of a sequencing-by-synthesis reaction by resequencing at least a portion of a nucleic acid template.
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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 nucleotide 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)
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11-12. -12. (canceled)
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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 the 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