title: Construction and analysis of randomized protein-encoding libraries using error-prone PCR.
abstract: In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.
authors.affiliation: Institute of Natural Sciences, Massey University, Auckland, New Zealand.
authors.forename: Paulina
authors.initials: P
authors.lastname: Hanson-Manful
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Patrick
mesh_terms: D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli; D015723:Gene Library; D005822:Genetic Vectors; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000235:genetics
chemical_list: D011506:Proteins