Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5375-5379, September 1980 Genetics Segment-directed mutagenesis: Construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule [site-specific mutagenesis/,-lactamase/recA protein/displacement-loop (D-loop) formation] DAVID SHORTLE*, DOUGLAS KOSHLAND*, GEORGE M. WEINSTOCKt, AND DAVID BOTSTEIN* *Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and tDepartment of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Communicated by D. Nathans, June 5,1980 ABSTRACI 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 P-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, Escberichia coli recA protein is used to catalyze assimilation of the homol- ogous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific SI 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- mu- tants 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 fragent 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 f-lac- tamase. Isolation of point mutations in a particular gene or regulatory site by random mutagenesis depends upon methods that will detect the rare mutant among a large background of nonmu- tants. When the mutant phenotype is unknown or difficult to discriminate from the wild type, acquisition of mutant alleles is often simply not feasible. Even when the gene has first been purified by recombinant DNA methods, considerable dif- ficulties remain: the gene is often not expressed in the cloned state, and assays for gene function may be too laborious to carry out on more than a few candidates. In the hope of improving the specificity and efficiency with which genes can be altered, several in vitro mutagenesis methods have been developed that generate base changes at predetermined sites on a DNA molecule (1-5). Two of these methods permit the construction of base substitutions in a small predetermined region of a duplex DNA molecule without the synthesis of unique oligonucleotides: mutagenesis by incorpo- ration of nucleotide analogues (1) and mutagenesis of small single-stranded gaps with sodium bisulfite (2). Both of these methods attain their site specificity by the deliberate placement of a single-strand interruption ("nick") in the region to be mutagenized. Site-specific nicking has been accomplished by using restriction endonucleases, which, under the appropriate conditions, nick rather than cleave their targets (2, 6). Nicks can be used directly as a site for mutagenesis; alternatively, the site The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5375 for mutagenesis can be moved a short distance to one side by limited nick-translation using DNA polymerase I of Escherichia coli (7). Nevertheless, the number of sites accessible to muta- genesis via restriction enzyme-catalyzed nicking is limited by the relative paucity of usable restriction targets. In this paper we describe a two-step enzymatic method that efficiently introduces nicks into any segment of a small circular DNA molecule, given only that the DNA of that segment can be isolated as a short single-stranded fragment. When coupled with local mutagenesis using the bisulfite procedure, this seg- ment-specific nicking procedure has allowed us to generate point mutations in two predetermined regions of the fl-lacta- mase gene carried by the small plasmid pBR322. MATERIALS AND METHODS Strains. The highly transformable E. colt strain HB101 (thr- leu- pro- recA- thi- hsdR- hsdM- supE ) and the small plasmid pBR322 (which confers resistance to ampicillin and tetracycline) were obtained from H. Boyer. The transformable Salmonella typhimurium strain DB4566 (hspL- hspS- proC9 his-2253 dhuAl purF145 galE503) was constructed by P22 transduction of strain TA3426 (same genotype plus bio- 203::TnlO; obtained from G. Ames) to biotin independence and tetracycline sensitivity. P22Ap31pfrl is a derivative of P22 that contains the struc- tural gene for (3-lactamase (the bla gene); its origin is described elsewhere (8). Deletion mutants of the bla gene made on pBR322 were transferred to strain DB4566 by transformation and then onto P22Ap31pfr1 by infecting these transformed derivatives and screening for bla recombinant phage. Deletion del2 was generated in vitro by cleaving pBR322 DNA with the restriction endonucleases Pvu I and Pst I, each of which cleave the plasmid once in the bla gene 128 base pairs (bp) apart. The digested plasmid was treated with S1 nuclease to produce blunt ends and cyclized with phage T4 DNA ligase (9). The approx- imate size of the deletion was confirmed by electrophoresis through agarose gels. The deletions de14391 and de14393 (kindly provided by K. Talmadge) extend from the Pst I site to codons 4 and 25, respectively, of the #-lactamase coding sequence, as determined by DNA sequence analysis (K. Tal- madge, personal communication). The relationships of these deletions and restriction sites are diagramed in Fig. 2 below. Enzymes. Restriction endonucleases Pvu I, Pst I, Taq I, and Hae III and E. coli exonuclease III were purchased from New England BioLabs; Micrococcus luteus DNA polymerase I and S1 nuclease were from Miles. All units of enzyme activity are those of the manufacturers. The recA protein of E. coli was purified to homogeneity by using a published procedure (10). Abbreviations: bp, base pair(s); D-loop, displacement loop.