Preparation of Templates for Nucleic Acid Sequencing

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.