Site-Directed Mutagenesis Using Altered /gb-Lactamase Specificity

Site-directed mutagenesis (SDM) is a powerful tool for the study of gene expression/regulation and protein structure and function. Hutchinson et al. (1 ) developed a general method for the introduction of specific changes in DNA sequence, which involves hybridization of a synthetic oligonucleotide (ON) containing the desired mutation to a single-stranded DNA (ssDNA) target template. Following hybridization, the oligonucleotide is extended with a DNA polymerase to create a double-stranded structure. The heteroduplex DNA is then transformed into an Escherichia coli , in which where both wild type and mutant strands are replicated. In the absence of any selection this method is very inefficient, often resulting in only a few percent of mutants obtained. Various strategies of selection have since been developed, which can increase mutagenesis efficiencies well above the theoretical yield of 50%. The methods of Kunkel (2 ), Eckstein (3 ), and Deng (4 ,5 ) employ negative selection against the wild-type DNA strand, in which the parental DNA is selectively degraded, either by growth in an alternate host strain, or by digestion with a nuclease or restriction enzyme. The methods of Lewis and Thompson (6 ) and Bonsack (7 ) utilize antibiotic resistance to positively select for the mutant DNA strand. This chapter describes a method for the positive selection of mutant strand DNA, which relies on the altered activity of the enzyme β-lactamase against extended spectrum cephalosporins (8 ).