Abstract
In vitro selection coupled with directed evolution represents a powerful method for generating nucleic acids and proteins with desired functional properties. Creating high-quality libraries of random sequences is an important step in this process as it allows variants of individual molecules to be generated from a single-parent sequence. These libraries are then screened for individual molecules with interesting, and sometimes very rare, phenotypes. Here, we describe a general method to introduce random nucleotide mutations into a parent sequence that takes advantage of the polymerase chain reaction (PCR). This protocol reduces mutational bias often associated with error-prone PCR methods and allows the experimenter to control the degree of mutagenesis by controlling the number of gene-doubling events that occur in the PCR reaction. The error-prone PCR method described here was used to optimize a de novo evolved protein for improved folding stability, solubility, and ligand-binding affinity.
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© 2010 Humana Press
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McCullum, E.O., Williams, B.A.R., Zhang, J., Chaput, J.C. (2010). Random Mutagenesis by Error-Prone PCR. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_7
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DOI: https://doi.org/10.1007/978-1-60761-652-8_7
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-651-1
Online ISBN: 978-1-60761-652-8
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