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

摘要: 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 multiple cloning site (MCS) flanked by two recognition sites identical cut sites, creating 5′-ATGC 5′-GCAT single-stranded ends. Any fragment into MCS could be amplified (i) excision BspMI, (ii) isolation, (iii) self-ligation of fragments using T4 ligase, (iv) selection multimers desired length, (v) them BspMI-digested original plasmid. Using this procedure, plasmids carrying either 30 copies 60-bp (a control experiment) or ten 1.2-kb luxA gene were constructed. The stable since repeat orientation, as determined restriction analysis. Potentially, not only but other class-IIS enzymes (with separated fixed distance from staggered points) applied, preferably those create 4-to-5-nucleotide-long utilize rather rare sites.