Consecutive cycles of precise, unidirectional 14-bp deletions using a BseRI/BsgI trimming plasmid

A straightforward method for generating precise, consecutive, unidirectional 14-bp deletions into cloned DNA, adopted from the trimming principle developed by Szybalski and his colleagues, is presented. The method utilizes pTRIM14, a plasmid constructed with the class-IIS restriction enzyme recognition sites for BseRI and BsgI arranged in the form of a cassette, just upstream from the cloned DNA. Class-IIS restriction enzymes cleave DNA downstream of their recognition sites. pTRIM14, containing the cloned DNA, is processed through a trimming cycle that involves sequential restriction digestions with BsgI and then BseRI, followed by treatment with Mung Bean nuclease and then with ligase. One trimming cycle results in a net 14-bp deletion. We demonstrate precise, consecutive deletions at very high efficiency.