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.
Huang
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Santi
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D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
D000596:Amino Acids; D003062:Codon; D009841:Oligonucleotides; D004247:DNA