earticle

영어

Lambda phage-originated recombination functions, collectively called Red, enable simple and specific in vivo gene manipulation in E. coli, which is of great use in functional genomics, BAC engineering and gene therapy. Directly from PCRs, linear dsDNAs flanked by short homology arms can be used as recombination substrates with high efficiency. However, their fundamental mechanisms remain to be scrutinized unambiguously for correct design of substrates and delicately-ramified applications. We examined the influence of terminally-linked non-homology on recombination functions, from which a strategy to show the validity of a ‘replication-dependent annealing’ model proposed previously was built: a linear dsDNA molecule harboring three homology region and two antibiotic resistance genes. Notably, its 3’ single stranded overhang complementary to lagging strand template of bacterial chromosome was most favorably annealed to the targets. Probably, it is caused by sequential exposure of chromosomal homology regions as the replication fork advances progressively, with lagging strand homology being unwound first. This bias was extensively observed throughout the chromosome, supporting the availability of the proposed model.