From Conventional Mutagenesis to Complex, Rationally-Design Mutant Libraries: Enabling New Opportunities in Scalability

Site Directed Mutagenesis (SDM), and specifically the QuikChange method has been an important feature of the research landscape with tens of thousands of research paper citations since its introduction in 1995. Continuing improvements to Pfu DNA polymerase have led to enhancements in the method, permitting protocols with higher fidelity, longer length-capability, multi-site targeting, and significantly faster workflows, (e.g., three times faster with QuikChange Lightning). New trends in mutagenesis seek to combine the advantages of random and sitedirected methods (that is: the ability to survey large target regions with no prior structural knowledge while enabling user-specified changes, and with the potential to develop all possible variants) in order to create, comprehensive, rationally designed mutant libraries. In this dynamic technological landscape, it is useful to survey how the features and benefits of the various methods have evolved and what this entails for the future of protein engineering.