Site-saturation mutagenesis by overlap extension PCR

Methods Mol Biol. 2014:1179:83-101. doi: 10.1007/978-1-4939-1053-3_6.


Site-saturation mutagenesis is a proven strategy for generating high-quality variant gene libraries of a defined size. Variation is introduced via incorporation of degenerate base combinations at specific codon locations, giving rise to a precise series of amino acid substitutions in the encoded protein. Here we describe a simple and efficient overlap PCR protocol for the introduction of degenerate bases at either single or multiple codon locations. The resulting libraries can then be directly screened for improved protein function as either an independent directed evolution study or an adjunct to random mutagenesis strategies (such as error-prone PCR) that are, in isolation, unlikely to access the full repertoire of possible amino acid substitutions at any given position.

MeSH terms

  • Mutagenesis / genetics
  • Mutagenesis / physiology*
  • Polymerase Chain Reaction / methods*