UNIT 26.6 Site-saturation Mutagenesis: A Powerful Tool for Structure-Based Design of Combinatorial Mutation Libraries

Abstract

This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants. The procedure accepts double-stranded plasmid isolated from the dam⁺ E. coli strain. The procedure is simple, fast, efficient, and eliminates time-consuming subcloning and ligation steps. Curr. Protoc. Protein Sci. 63:26.6.1-26.6.10. © 2011 by John Wiley & Sons, Inc.