Use of Codon Cassette Mutagenesis for Saturation Mutagenesis

Cassette mutagenesis methods that introduce site-specific sequence changes into a target gene are powerful tools for the manipulation of proteins to analyze then structure and function (1 –3 ). Typically, a small target region is excised by cleavage at two constructed or naturally occurring restriction enzyme sites that flank this region, and the excised portion is replaced with oligonucleotides containing the desired mutation. In general, this method is used to mutagenize regions bracketed by closely spaced cleavage sites for restriction endonucleases that do not cleave elsewhere in the target plasmid. Use of cassette and other site-directed mutagenesis methods for saturation of one or more positions in a protein with all possible amino acid substitutions requires the costly synthesis of many customized oligonucleotides both to generate the necessary cleavage sites and to introduce each desired amino acid change. Much of the cost of oligonucleotide synthesis can be avoided by using degenerate oligonucleotides that have the potential to create several different mutations (4 ,5 ). However, the use of degenerate oligonucleotides often requires substantial screening efforts because of the difficulty in identifying the final few nucleotide changes required to complete the set of desired mutants.