Segment-directed mutagenesis: Construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the β-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ('D-loop') in the circular DNA. Second, a small amount of the single-strand specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla^- mutants and 49/51 of the mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the 'signal' sequence thought to be involved in secretion of β-lactamase.