Patent ID: 8304189

Claim:
A method for sequencing all or part of a nucleic acid segment, the method comprising: (a) providing a first nucleic acid molecule comprising the nucleic acid segment flanked by a first attP recombination site and a second attP recombination site which can not recombine with each other and wherein the first and the second attP recombination sites each have a sequencing primer binding site located within 30 nucleotides of the nucleic acid segment wherein the sequencing primer binding sites encompass the IHF site of the first and the second attP site and wherein the sequencing primer binding sites differ from each other by one, two, three or four nucleotides, (b) providing a second nucleic acid molecule comprising a first attB recombination site and a second attB recombination site which can not recombine with each other, (c) combining the first and the second nucleic acid molecules in the presence of one or more recombinases under conditions which allow for site specific recombination in trans between, (1) the first attP recombination site and the first attB recombination site and, (2) the second attP recombination site and the second attB recombination site, such that a product nucleic acid molecule is generated which contains the nucleic acid segment flanked by a first and a second attL recombination sites, wherein the product attL recombination sites comprise the unique binding sites encompassing the IHF site from the attP recombination sites, (d) sequencing all or part of a first strand of the product nucleic acid molecules comprising the attL sites with a sequencing primer which mediates 5′ to 3′ extension from the unique sequencing primer binding site located within the first attL recombination site, and (e) sequencing all or part of a second strand of the nucleic acid segment comprising the attL sites with a sequencing primer which mediates 5′ to 3′ extension from the sequencing primer binding site located within the second attL site specific recombination site.