Random mutagenesis by error-prone PCR.
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.
McCullum
Elizabeth O
EO
The Biodesign Institute, and Department of Chemistry and Biochemistry, Center for BioOptical Nanotechnology, Arizona State University, Tempe, AZ, USA.
Williams
Berea A R
BA
Zhang
Jinglei
J
Chaput
John C
JC
D019020:Directed Molecular Evolution; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*