Preparation of templates for nucleic acid sequencing

摘要

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.

主权项

1. A method of preparing single-stranded templates for a nucleic acid sequencing reaction comprising,
(i) providing a solid support comprising a plurality of amplification primers, wherein a subset of said plurality of amplification primers comprises a cleavage site; (ii) amplifying a template using subset of the primers on the support to produce a plurality of double-stranded nucleic acid molecules, wherein both strands of each double-stranded nucleic acid molecule are attached to the solid support at their 5′ ends, whereby the cleavage site is positioned in a double-stranded region of each double-stranded molecule; (iii) cleaving only one strand of the double stranded molecules at the cleavage site; (iv) subjecting the cleaved strand to denaturing conditions to remove the portion of the cleaved strand not attached to the solid support, followed by re-annealing the cleaved strand to the opposite strand, thereby generating immobilized partially or substantially single-stranded templates; and (v) hybridizing a sequencing primer to the immobilized partially or substantially single-stranded templates, thereby preparing single-stranded templates for a nucleic acid sequencing reaction.