Patent ID: 9085802
Filing Date: 2015-07-21
Classification: B01J,C12Q

Abstract:
1. A method of preparing single-stranded templates for a nucleic acid sequencing reaction comprising, (i) providing a solid support comprising a plurality amplification primers, wherein a subset of said plurality of amplification primers comprises a cleavage site; (ii) amplifying a template using the subset of 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, 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, 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.