Preparation of Templates for Nucleic Acid Sequencing
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Abstract
The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise:
- providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end,
- cleaving one or both strands of the double-stranded nucleic acid molecule, and
- subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction,
4 Citations
76 Claims
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1-56. -56. (canceled)
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57. A method of generating a template for a nucleic acid sequencing reaction comprising,
(i) providing a solid support comprising a plurality of forward amplification primers and a plurality of reverse amplification primers, wherein said plurality of forward amplification primers or said plurality of reverse amplification primers comprises a cleavage site; -
(ii) amplifying a template using the primers on the support to produce a plurality of double-stranded nucleic acid molecules, wherein both strands of the molecule are attached to the solid support at their 5′
ends;(iii) cleaving one strand of the double stranded molecules at the cleavage site, the cleavage not comprising cleavage with a restriction endonuclease or a nicking endonuclease; (iv) subjecting the cleaved strand to denaturing conditions to remove the portion of the cleaved strand not attached to the solid support, thereby generating partially or substantially single-stranded immobilized single-stranded templates immobilized to the solid support; and (v) hybridizing a sequencing primer to the immobilized single-stranded templates. - View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)
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Specification