SEQUENCING METHODS AND COMPOSITIONS
First Claim
1. A method for obtaining sequence information from a portion of a nucleic acid template, comprising:
- hybridizing a first primer to a template strand;
sequencing a portion of the nucleic acid template, wherein the sequencing includes extending the first primer, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template;
introducing a nick into a portion of the template strand that is hybridized to the extended first primer product;
wherein the nick includes a free 5′
end and a free 3′
end in the template strand;
degrading a portion of the template strand from the free 5′
end of the nick using a degrading agent, thereby generating a single-stranded portion of the extended first primer product, wherein a portion of the extended first primer product remains hybridized to an undegraded portion of the template strand; and
sequencing at least some of the single-stranded portion of the extended first primer product.
1 Assignment
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Accused Products
Abstract
Provided herein are compositions, systems, methods and kits useful for obtaining sequence information from a nucleic acid molecule. In some embodiments, the compositions, systems, methods and kits are useful for sequencing of natural or modified nucleic acids. In some embodiments, the compositions, systems, methods and kits relate to bi-directional sequencing of nucleic acids. In some embodiments, the compositions, systems, methods and kits relate to sequencing of nucleic acids linked to a solid support. In some embodiments, the methods useful for obtaining sequence information from a nucleic acid molecule include label-free or ion-based sequencing methods.
69 Citations
27 Claims
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1. A method for obtaining sequence information from a portion of a nucleic acid template, comprising:
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hybridizing a first primer to a template strand; sequencing a portion of the nucleic acid template, wherein the sequencing includes extending the first primer, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template; introducing a nick into a portion of the template strand that is hybridized to the extended first primer product;
wherein the nick includes a free 5′
end and a free 3′
end in the template strand;degrading a portion of the template strand from the free 5′
end of the nick using a degrading agent, thereby generating a single-stranded portion of the extended first primer product, wherein a portion of the extended first primer product remains hybridized to an undegraded portion of the template strand; andsequencing at least some of the single-stranded portion of the extended first primer product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for nucleic acid sequencing comprising:
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hybridizing a first primer to a distal end of a nucleic acid strand having a distal and proximal end, wherein the proximal end of the nucleic acid strand is linked to a support; extending the hybridized first primer in the direction of the proximal end of the nucleic acid strand and the support, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid strand and obtaining a first sequencing read; introducing a site-specific nick into the proximal end of the nucleic strand hybridized to the extended first primer product; degrading a portion of the nucleic acid strand, thereby generating a single-stranded portion within the extended first primer product, wherein a portion of the extended first primer product remains hybridized to the nucleic acid strand; and extending the single-stranded portion within the extended first primer product, thereby obtaining a second sequencing read. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for obtaining bi-directional sequence information from a nucleic acid molecule, comprising:
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hybridizing a first primer to a nucleic acid molecule; sequencing a portion of the nucleic acid molecule, wherein the sequencing includes extending the first primer, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid molecule; introducing a nick into a portion of the nucleic acid molecule that is hybridized to the extended first primer product;
wherein the nick includes a free 5′
end and a free 3′
end in the nucleic acid molecule;degrading a portion of the nucleic acid molecule from the free 5′
end of the nick using a degrading agent, thereby generating a single-stranded portion of the extended first primer product, wherein a portion of the extended first primer product remains hybridized to an undegraded portion of the nucleic acid molecule; andsequencing at least some of the single-stranded portion of the extended first primer product. - View Dependent Claims (24, 25, 26)
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27. A method for improving nucleic acid sequencing accuracy comprising:
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hybridizing a first primer to a distal end of a nucleic acid strand having a distal and proximal end, wherein the proximal end of the nucleic acid strand is linked to a solid support; extending the hybridized first primer in the direction of the proximal end of the nucleic acid strand and the solid support, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid strand and obtaining a first sequencing read; introducing a site-specific nick into the proximal end of the nucleic strand hybridized to the extended first primer product; degrading a portion of the nucleic acid strand, thereby generating a single-stranded portion within the extended first primer product, wherein a portion of the extended first primer product remains hybridized to the nucleic acid strand; and extending the single-stranded portion within the extended first primer product, thereby obtaining a second sequencing read; and aligning the first and second sequencing read to obtain a nucleic acid sequence with improved accuracy.
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Specification