Abstract
Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lutz S (2010) Beyond directed evolution—semi-rational protein engineering and design. Curr Opin Biotechnol 21(6):734–743
Chica RA, Doucet N, Pelletier JN (2005) Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr Opin Biotechnol 16(4):378–384
Lutz S, Patrick WM (2004) Novel methods for directed evolution of enzymes: quality, not quantity. Curr Opin Biotechnol 15(4):291–297
Barak Y, Ackerley DF, Dodge CJ, Banwari L, Alex C, Francis AJ, Matin A (2006) Analysis of novel soluble chromate and uranyl reductases and generation of an improved enzyme by directed evolution. Appl Environ Microbiol 72(11):7074–7082
Morley KL, Kazlauskas RJ (2005) Improving enzyme properties: when are closer mutations better? Trends Biotechnol 23(5):231–237
Hermes JD, Blacklow SC, Knowles JR (1990) Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme. Proc Natl Acad Sci U S A 87(2):696–700
Horsman GP, Liu AM, Henke E, Bornscheuer UT, Kazlauskas RJ (2003) Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl-3-phenylbutyrate. Chemistry 9(9):1933–1939
Kumar S, Chen CS, Waxman DJ, Halpert JR (2005) Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide. J Biol Chem 280(20):19569–19575
Wong JT (1980) Role of minimization of chemical distances between amino acids in the evolution of the genetic code. Proc Natl Acad Sci U S A 77(2):1083–1086
Patrick WM, Firth AE, Blackburn JM (2003) User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. Protein Eng 16:451–457
Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11–15
Cadwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2(1):28–33
Fromant M, Blanquet S, Plateau P (1995) Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal Biochem 224(1):347–353
Lin-Goerke JL, Robbins DJ, Burczak JD (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques 23(3):409–412
Rasila TS, Pajunen MI, Savilahti H (2009) Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment. Anal Biochem 338(1):71–80
Hanson-Manful P, Patrick WM (2013) Construction and analysis of randomized protein-encoding libraries using error-prone PCR. Methods Mol Biol 996:251–267
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY
Saraswat M, Grand RS, Patrick WM (2013) Desalting DNA by drop dialysis increases library size upon transformation. Biosci Biotechnol Biochem 77(2):402–404
Firth AE, Patrick WM (2008) GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries. Nucleic Acids Res 36:W281–W285
Hartman PS (1991) Transillumination can profoundly reduce transformation frequencies. Biotechniques 11:747–748
Goldsmith M, Kiss C, Bradbury AR, Tawfik DS (2007) Avoiding and controlling double transformation artifacts. Protein Eng Des Sel 20(7):315–318
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Copp, J.N., Hanson-Manful, P., Ackerley, D.F., Patrick, W.M. (2014). Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1053-3_1
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-1052-6
Online ISBN: 978-1-4939-1053-3
eBook Packages: Springer Protocols