Chapter title |
In Vitro Mutagenesis Protocols
|
---|---|
Chapter number | 7 |
Book title |
In Vitro Mutagenesis Protocols
|
Published in |
Methods in molecular biology, January 2010
|
DOI | 10.1007/978-1-60761-652-8_7 |
Pubmed ID | |
Book ISBNs |
978-1-60761-651-1, 978-1-60761-652-8
|
Authors |
McCullum, Elizabeth O, Williams, Berea A R, Zhang, Jinglei, Chaput, John C, Elizabeth O. McCullum, Berea A. R. Williams, Jinglei Zhang, John C. Chaput, McCullum, Elizabeth O., Williams, Berea A. R., Chaput, John C. |
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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