Combinatorial libraries of proteins: analysis of efficiency of mutagenesis techniques.

A number of considerations are made on the efficiency of mutagenesis techniques used in protein engineering, particularly those that include a random component. The expected outcome of different protocols is analyzed using computer programs. Special emphasis is made on the effect that the degeneracy of the genetic code has on the bias of the representation of amino acid replacements. The consequences of using alternative methods is analyzed in terms of the likelihood of obtaining underrepresented amino acid substitutions in mutant libraries. A consideration is also made of the outcome of combinatorial mutagenesis experiments with regard to the size of the amino acid window and the multiplicity of replacements that could be sampled with different methods. Optimal mutagenesis rates for specific conditions could be derived from the presented data and the computer program made available with this paper.