Abstract
Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747–10751, 1994; Stemmer, Nature 370, 389–391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.
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Acknowledgments
This study was supported by the High Technology Research and Development Program of China (2006AA02Z221) and the National Natural Science Foundation of China (31171204) to H.F.
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Feng, H., Wang, HY., Zhao, HY. (2017). Novel Random Mutagenesis Method for Directed Evolution. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_32
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DOI: https://doi.org/10.1007/978-1-4939-6472-7_32
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