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. How ever, the presence of the wild-type codon in the degenerate codon pres ents some inconvenience in screening and identification of catalytical ly active mutants. The wild-type enzyme will always be identified as c atalytically 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 identifica tion of active mutants. This paper describes a simple approach for exc lusion of the wild-type codon in degenerate codons through the synthes is of two or three oligonucleotide mixtures. The minimum number of ind ividual colonies required to achieve a high degree of certainty of inc luding 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 proces s and saves time and expense in DNA sequencing. (C) 1994 Academic Pres s, Inc.