Advertisement

DNA Shuffling

  • John M. Joern
Part of the Methods in Molecular Biology™ book series (MIMB, volume 231)

Abstract

DNA shuffling is a method for in vitro recombination of homologous genes invented by W.P.C Stemmer (1). The genes to be recombined are randomly fragmented by DNaseI, and fragments of the desired size are purified from an agarose gel. These fragments are then reassembled using cycles of denaturation, annealing, and extension by a polymerase (see Fig. 1). Recombination occurs when fragments from different parents anneal at a region of high sequence identity. Following this reassembly reaction, PCR amplification with primers is used to generate full-length chimeras suitable for cloning into an expression vector.
Fig. 1.

Schematic of DNA shuffling method. Parental genes are cleaved randomly using DNaseI to generate a pool of fragments. These fragments are recombined using PCR with a specialized thermocycling protocol. Fragments are denatured at high temperature, then allowed to anneal to other fragments. Some of these annealing events result in heteroduplexes of fragments from two homologous parents. Annealed 3′ ends are then extended by polymerase. After 20–50 cycles of assembly, a PCR amplification with primers is used to selectively amplify full-length sequences.

Keywords

Outer Primer Manganese Chloride Parent Anneal Mutagenesis Rate Desired Size Range 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Stemmer, W. P. C. (1994) DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10,747–10,751.PubMedCrossRefGoogle Scholar
  2. 2.
    Chang, C. J., Chen, T. T., Cox, B. W., et al. (1999) Evolution of a cytokine using DNA family shuffling. Nat. Biotechnol. 17, 793–797.PubMedCrossRefGoogle Scholar
  3. 3.
    Ness, J. E., Welch, M., Giver, L., et al. (1999) DNA shuffling of subgenomic sequences of subtilisin. Nat. Biotechnol. 17, 893–896.PubMedCrossRefGoogle Scholar
  4. 4.
    Christians, F. C., Scapozza, L., Crameri, A., Folkers, G. and Stemmer, W. P. C. (1999) Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. Nat. Biotechnol. 17, 259–264.PubMedCrossRefGoogle Scholar
  5. 5.
    Bruhlmann, F. and Chen, W. (1998) Tuning biphenyl dioxygenase for extended substrate specificity. Biotech. Bioeng. 63, 544–551.CrossRefGoogle Scholar
  6. 6.
    Zhao, H. and Arnold, F. H. (1997) Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25, 1307–1308.PubMedCrossRefGoogle Scholar
  7. 7.
    Kikuchi, M., Ohnishi, K., and Harayama, S. (1999) Novel family shuffling methods for the in vitro evolution of enzymes. Gene 236, 159–167.PubMedCrossRefGoogle Scholar
  8. 8.
    Joern, J. M., Meinhold, P., and Arnold, F. H. (2002) Analysis of Shuffled Gene Libraries. J. Mol. Biol. 316, 643–656.PubMedCrossRefGoogle Scholar
  9. 9.
    Abècassis, 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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • John M. Joern
    • 1
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadena

Personalised recommendations