Amplification of cloned DNA as tandem multimers using BspMI-generated asymmetric cohesive ends

Abstract

By generating totally asymmetric and complementary cohesive ends it is possible to amplify any cloned DNA fragment, while assuring that all repeating units are ligated in the same orientation. Starting with plasmid pUC18, which contains a unique BspMI site, an amplification plasmid, pSK3, was constructed in which a multiple cloning site (MCS) is flanked by two BspMI recognition sites with identical cut sites, creating the complementary 5′-ATGC and 5′-GCAT single-stranded ends. Any DNA fragment cloned into the MCS could be amplified by (i) excision with BspMI, (ii) fragment isolation, (iii) self-ligation of the fragments using T4 DNA ligase, (iv) selection of multimers of desired length, and (v) cloning them into the BspMI-digested original plasmid. Using this procedure, plasmids carrying either 30 copies of the 60-bp MCS fragment (a control experiment) or ten copies of the 1.2-kb luxA gene fragment were constructed. The plasmids were stable since all the repeat units were in the same orientation, as determined by restriction analysis. Potentially, not only BspMI but other class-IIS restriction enzymes (with recognition sites separated by a fixed distance from the staggered cut points) could be applied, preferably those that create 4-to-5-nucleotide-long cohesive ends and utilize rather rare recognition sites.