title: Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.
abstract: 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 beta-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 these 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 beta-lactamase.
mesh_terms: D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D008836:Micrococcal Nuclease; D009154:Mutation; D010957:Plasmids; D013447:Sulfites / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D004270:DNA, Circular; D004277:DNA, Single-Stranded; D013447:Sulfites; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease; D001618:beta-Lactamases; C009279:sodium bisulfite