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Updated: Aug 14, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Generation of multiple independent substitution mutants by M13 in vitro mutagenesis using a single mutagenic
1Division of Basic Research, Children's Hospital Research Foundation, Cincinnati, OH 45229.
Abstract:
A 56-nucleotide mutagenic oligomer containing six mismatches with the wild-type template was used to construct multiple transversion mutations in the putative heparin binding region of the rat neural cell adhesion molecule (NCAM) cDNA sequence. Mutants were screened by hybridization to the 56-mer. The relative stability of a mutant DNA:56-mer duplex correlated with the number of base substitutions present in the mutant sequence. Five independent categories of mutants carrying from two to five of the expected nucleotide substitutions were isolated. No mutations other than those directed by the 56-mer were observed. These results suggest a method for generating sets of related predefined substitution mutants.
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