Cassette mutagenesis involves replacing a wild-type DNA sequence with synthetic double-stranded oligonucleotides in order to introduce one or more mutations (1-6). The technique allows saturation of a target amino acid codon with mutations, and can be used to probe the role of that particular residue in a protein and the effect of specific point mutations in it. In their description of the method, Wells et al. (4) synthesized single-stranded oligonucleotides containing different codons over the target in separate pools. Oligonucleotide-site-directed mutagenesis procedures are used to generate restriction sites that closely flank the target codon in the plasmid containing the gene of interest. Use of the appropriate restriction endonucleases permits insertion of the synthetic duplex cassettes. These are designed to restore fully the wild-type coding sequence except over the target codon, and also to eliminate one of the restriction sites. This latter point is important for selection of the clones containing the mutant cassette.
Several variations on the original technique have been developed for specific purposes. For example, Reidhaar-Olsen and Sauer (5) described a combinatorial method, in which they randomized two or three positions by oligonucleotide cassette mutagenesis, selected for functional protein and then sequenced to determine the spectrum of allowable substitutions at each position. They applied the method repeatedly in order to examine the role of different substitutions on the DNA-binding domain of lambda repressor. Kegler-Ebo et al. (6) described codon cassette mutagenesis as a way of depositing single codons at specific sites in double-stranded DNA. Using this method, a series of 11 cassettes is sufficient to insert all possible amino acids at any constructed target site.
