Methods for sequencing nucleic acids
First Claim
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1. A method for sequencing a template nucleic acid molecule, comprising:
- (a) contacting a stretched and primed template nucleic acid molecule with a substrate, the primed template nucleic acid molecule comprising one or more primer-binding sites, the substrate comprising primers immobilized thereon,each primer comprising;
(i) a region complementary to a primer-binding site, and(ii) a barcode sequence indicative of a physical position of the primer on the substrate;
(b) conducting extension reactions using the primers and the template nucleic acid molecule as a template, thereby generating extension products, each extension product comprises (i) sequence of a fragment of the template nucleic acid or complement of the fragment, and (ii) sequence of the barcode sequence or a complement thereof;
(c) sequencing the extension products to determine sequences of the fragments or complements thereof and barcode sequences or complements thereof; and
(d) assembling the sequences of the fragments using the barcode sequence to thereby determine sequence of the template nucleic acid molecule.
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Abstract
Provided herein are methods and compositions for the sequencing of long nucleic acids, such as DNA. The methods and compositions are suited for the spatial labeling and sequencing of long nucleic acid molecules.
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Citations
20 Claims
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1. A method for sequencing a template nucleic acid molecule, comprising:
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(a) contacting a stretched and primed template nucleic acid molecule with a substrate, the primed template nucleic acid molecule comprising one or more primer-binding sites, the substrate comprising primers immobilized thereon, each primer comprising; (i) a region complementary to a primer-binding site, and (ii) a barcode sequence indicative of a physical position of the primer on the substrate; (b) conducting extension reactions using the primers and the template nucleic acid molecule as a template, thereby generating extension products, each extension product comprises (i) sequence of a fragment of the template nucleic acid or complement of the fragment, and (ii) sequence of the barcode sequence or a complement thereof; (c) sequencing the extension products to determine sequences of the fragments or complements thereof and barcode sequences or complements thereof; and (d) assembling the sequences of the fragments using the barcode sequence to thereby determine sequence of the template nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for determining sequence of a template nucleic acid molecule, comprising:
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(a) contacting the template nucleic acid molecule with a plurality of probes immobilized on a surface of a nucleic acid probe array, each the probe being uniquely tagged based on its location on the nucleic acid probe array; (b) performing extension reactions at locations of probe;
template contact points on the template nucleic acid molecule; and(c) using the tags to compile the sequence of the template nucleic acid molecule, wherein the method further comprises stretching the template nucleic acid molecule prior to (a). - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
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Specification