Patent ID: 8715966

Claim:
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 one or more methylated nucleotides; (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 molecules are attached to the solid support at their 5′ ends, thereby producing a hemimethylated duplex structure in each double-stranded nucleic acid molecule; (iii) cleaving one strand of the double stranded molecules at said hemimethylated duplex structure with an endonuclease enzyme specific for a recognition sequence including the one or more methylated nucleotides; (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 templates for a nucleic acid sequencing reaction.