In Vitro Mutagenesis Protocols

McCullum, Elizabeth O, Williams, Berea A R, Zhang, Jinglei, Chaput, John C, Elizabeth O. McCullum, Berea A. R. Williams, Jinglei Zhang, John C. Chaput, McCullum, Elizabeth O., Williams, Berea A. R., Chaput, John C.

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.