Identification of biologically active mutants by combinatorial cassette mutagenesis: exclusion of wild-type codon from degenerate codons.

A degenerate codon (N)(N)(G+C) is often used in cassette mutagenesis to encode all 20 natural amino acids at the target mutation site. However, the presence of the wild-type codon in the degenerate codon presents some inconvenience in screening and identification of catalytically active mutants. The wild-type enzyme will always be identified as catalytically active in a screening process and in most cases can only be distinguished from active mutants by DNA sequencing. Sequencing of background wild-type enzyme represents wasted effort in the identification of active mutants. This paper describes a simple approach for exclusion of the wild-type codon in degenerate codons through the synthesis of two or three oligonucleotide mixtures. The minimum number of individual colonies required to achieve a high degree of certainty of including all possible codons for screening of catalytic activity can be estimated using a statistical procedure. The use of degenerate codons that exclude the wild-type amino acid facilitates the screening process and saves time and expense in DNA sequencing.