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.
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Acknowledgments
Financial support by the Defense Threat Reduction Agency (DTRA) of the US Department of Defense (contract HDTRA1-11-C-0026) and by the CounterACT Program, National Institutes of Health Office of the Director, and the National Institute of Neurological Diseases and Stroke (Grant Number U54-NS058183-06) are gratefully acknowledged.
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Rockah-Shmuel, L., Tawfik, D.S., Goldsmith, M. (2014). Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides During Gene Shuffling (ISOR). In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_8
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DOI: https://doi.org/10.1007/978-1-4939-1053-3_8
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