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, BspMI, 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 BspMI 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 BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of (i) BspMI 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 BspMI 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.