Random mutagenesis by error-prone PCR

Elizabeth O. McCullum, Berea A.R. Williams, Jinglei Zhang, John C. Chaput

Research output: Chapter in Book/Report/Conference proceedingChapter

67 Scopus citations

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 ptimize ade novo evolved protein for improved folding stability, solubility, and ligand-binding affinity.

Original languageEnglish (US)
Title of host publicationIn Vitro Mutagenesis Protocols
Subtitle of host publicationThird Edition
EditorsBraman Jeff
Pages103-109
Number of pages7
DOIs
StatePublished - Dec 1 2010

Publication series

NameMethods in Molecular Biology
Volume634
ISSN (Print)1064-3745

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Keywords

  • Directed evolution
  • Error-prone PCR
  • Taq DNA polymerase

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

McCullum, E. O., Williams, B. A. R., Zhang, J., & Chaput, J. C. (2010). Random mutagenesis by error-prone PCR. In B. Jeff (Ed.), In Vitro Mutagenesis Protocols: Third Edition (pp. 103-109). (Methods in Molecular Biology; Vol. 634). https://doi.org/10.1007/978-1-60761-652-8_7