title: Random mutagenesis by error-prone PCR.
abstract: In vitro selection coupled with directed evolution represents a powerful method for generating nucleic acids and proteins with desired functional properties. Creating high-quality libraries of random sequences is an important step in this process as it allows variants of individual molecules to be generated from a single-parent sequence. These libraries are then screened for individual molecules with interesting, and sometimes very rare, phenotypes. Here, we describe a general method to introduce random nucleotide mutations into a parent sequence that takes advantage of the polymerase chain reaction (PCR). This protocol reduces mutational bias often associated with error-prone PCR methods and allows the experimenter to control the degree of mutagenesis by controlling the number of gene-doubling events that occur in the PCR reaction. The error-prone PCR method described here was used to optimize a de novo evolved protein for improved folding stability, solubility, and ligand-binding affinity.
authors.affiliation: The Biodesign Institute, and Department of Chemistry and Biochemistry, Center for BioOptical Nanotechnology, Arizona State University, Tempe, AZ, USA.
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: McCullum
authors.forename: Berea A R
authors.initials: BA
authors.lastname: Williams
authors.forename: Jinglei
authors.initials: J
authors.lastname: Zhang
authors.forename: John C
authors.initials: JC
authors.lastname: Chaput
mesh_terms: D019020:Directed Molecular Evolution; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*