Construction and Analysis of Randomized Protein-Encoding Libraries Using Error-Prone PCR

  • Paulina Hanson-Manful
  • Wayne M. Patrick
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 996)

Abstract

In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.

Key words

Directed evolution Random mutagenesis Error-prone PCR GeneMorph II Mutazyme II DNA polymerase Library Mutation spectrum Mutational bias PEDEL-AA 

Notes

Acknowledgment

The authors gratefully acknowledge financial support for this work from the New Zealand Marsden Fund.

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Paulina Hanson-Manful
    • 1
  • Wayne M. Patrick
    • 2
    • 1
  1. 1.Institute of Natural SciencesMassey UniversityAucklandNew Zealand
  2. 2.Department of BiochemistryUniversity of OtagoDunedinNew Zealand

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