A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

Abstract A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, Bsp MI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3′ from its recognition site 5′-ACCTGC-3′. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique Bsp MI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the Bsp MI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of (i) Bsp MI cleavage, (ii) removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and (iii) blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZα . gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process. The partial fill-in of the cohesive ends with less than four deoxyribonucleotide triphosphates, as mediated by Klenow fragment of E. coli DNA polymerase I (PolIk), would result in 1, 2 or 3-bp deletions or additions in a given cleavage cycle, which permits one to create precise deletions. An analogous series of cycles of Bsp MI cleavage employing a PolIk-mediated fill-in reaction instead of the MB digestion step, and followed by ligation, would permit one to synthesize various 4-bp direct-repeat sequences and desired variants of them.