Library Generation by Gene Shuffling

Adam J. Meyer1, Jared W. Ellefson1, Andrew D. Ellington1

1 University of Texas at Austin, Austin, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.12
DOI:  10.1002/0471142727.mb1512s105
Online Posting Date:  January, 2014
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This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer‐less PCR. The resulting chimeric genes can then be screened or selected for a desired function. Curr. Protoc. Mol. Biol. 105:15.12.1‐15.12.7. © 2014 by John Wiley & Sons, Inc.

Keywords: directed evolution; recombination; PCR

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Parental plasmid DNA (see Generation of parental plasmid DNA in the Critical Parameters section)
  • 1 M Tris·Cl, pH 7.4 ( appendix 22)
  • 200 mM MnCl 2
  • DNase I
  • Distilled water
  • PCR clean‐up kit (e.g., Promega Wizard SV Gel and PCR Clean‐Up System)
  • DNA Polymerase Family A and Family B Blend (see Polymerase choices in the Critical Parameters section)
  • 10 µl 600 mM Tris‐SO 4 (pH 8.9), 180 mM ammonium sulfate
  • 4 mM dNTPs
  • 50 mM MgSO 4
  • Thermostable, proofreading DNA polymerase (e.g., Pfu DNA polymerase or KOD DNA polymerase) and associated buffer (see Polymerase choices in the Critical Parameters section)
  • 20 µM inner primers (See Critical Parameter‐Primer Design and Fig. )
  • 20 µM outer primers or restriction enzymes (See Critical Parameters‐Primer Design and Fig. )
  • 0.2‐ml PCR tubes
  • Thermal cycler (see unit 15.1)
  • Additional reagents and equipment for PCR amplification (unit 15.1) and agarose gel electrophoresis (unit 2.7)
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Literature Cited

Literature Cited
  Abécassis, V., Pompon, D., and Truan, G. 2000. High efficiency family shuffling based on multi‐step PCR and in vivo DNA recombination in yeast: Statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res. 28:E88.
  Bershtein, S., Goldin, K., and Tawfik, D.S. 2008. Intense neutral drifts yield robust and evolvable consensus proteins. J. Mol. Biol. 379:1029‐1044.
  Brühlmann, F. and Chen, W. 1999. Tuning biphenyl dioxygenase for extended substrate specificity. Biotechnol. Bioeng. 63:544‐551.
  Farrow, M.F. and Arnold, F.H. 2010. Combinatorial recombination of gene fragments to construct a library of chimeras. Curr. Protoc. Prot. Sci. 26.2.1‐26.2.20.
  Giver, L., Gershenson, A., Freskgard, P.O., and Arnold, F.H. 1998. Directed evolution of a thermostable esterase. Proc. Natl. Acad. Sci. U.S.A. 95:12809‐12813.
  Hiraga, K. and Arnold, F.H. 2003. General method for sequence‐independent site‐directed chimeragenesis. J. Mol. Biol. 330:287‐296.
  Jézéquel, L., Loeper, J., and Pompon, D. 2008. Sequence‐independent construction of ordered combinatorial libraries with predefined crossover points. BioTechniques 45:523‐532.
  Joern, J.M., Meinhold, P., and Arnold, F.H. 2002. Analysis of shuffled gene libraries. J. Mol. Biol. 316:643‐656.
  Lorimer, I.A. and Pastan, I. 1995. Random recombination of antibody single chain Fv sequences after fragmentation with DNaseI in the presence of Mn2+. Nucleic Acids Res. 23:3067‐3068.
  Maheshri, N., Koerber, J.T., Kaspar, B.K., and Schaffer, D.V 2006. Directed evolution of adeno‐associated virus yields enhanced gene delivery vectors. Nat. Biotechnol. 24:198‐204.
  Miyazaki, K. and Arnold, F.H. 1999. Exploring nonnatural evolutionary pathways by saturation mutagenesis: Rapid improvement of protein function. J. Mol. Evol. 49:716‐720.
  Pekrun, K., Shibata, R., Igarashi, T., Reed, M., Sheppard, L., Patten, P.A., Stemmer, W.P.C., Martin, M.A., and Soong, N.‐W. 2002. Evolution of a human immunodeficiency virus type 1 variant with enhanced replication in pig‐tailed macaque cells by DNA shuffling. J. Virol. 76:2924‐2935.
  Stemmer, W.P. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389‐391.
  Zhao, H. and Arnold, F.H. 1997. Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25:1307‐1308.
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