Patent ID: 8017335

Claim:
A method for pairwise sequencing of first and second regions of a target double-stranded polynucleotide, wherein said first and second regions are in complementary strands of the target polynucleotide, the method comprising: (a) providing a solid support having immobilised thereon a plurality of template polynucleotide duplexes each comprising the double-stranded target polynucleotide, wherein each template duplex is formed from complementary first and second template strands linked to the solid support at their 5′ ends; (b) cleaving the second template strands of a sub-fraction of the template polynucleotide duplexes to remove all or a portion of said strands, thereby generating single-stranded regions on the complementary first template strands; (c) hybridising first sequencing primers to the single-stranded regions of the first template strands generated in part (b); (d) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primer to determine the sequence of a first region of the target polynucleotide in the first template strand; (e) cleaving the first template strands of substantially all intact template polynucleotide duplexes not cleaved in step (b) to remove all or a portion of said strands, thereby generating single-stranded regions on the second template strands that were not cleaved in step (b); (f) hybridising a second sequencing primer to the single-stranded regions of the second template strands generated in part (e); and (g) carrying out a second sequencing reaction by sequential addition of nucleotides to the second sequencing primer to determine the sequence of a second region of the target polynucleotide in the second template strand.