Generating targeted libraries by the combinatorial incorporation of synthetic oligonucleotides during gene shuffling (ISOR)

Abstract

Protein engineering by directed evolution relies on the use of libraries enriched with beneficial variants. Such libraries should explore large mutational diversities while avoiding high loads of deleterious mutations. Here we describe a simple protocol for incorporating synthetic oligonucleotides that encode designed, site-specific mutations by assembly PCR. This protocol enables a researcher to "hedge the bets," namely, to explore a large number of potentially beneficial mutations in a combinatorial manner such that individual library variants carry a limited number of mutations. © 2014 Springer Science+Business Media New York.