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DNA shuffling: Modifying the hand that nature dealt

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Summary

DNA shuffling is a technique being utilized for in vitro recombination of a single gene or pools of homologous genes. The genes are fragmented into randomly sized pieces, and polymerase chain reaction (PCR) reassembly of full-length genes from the fragments, via self-priming, yields recombination due to PCR template switching. After these PCR products are screened and the interesting products sequenced, improved clones are reshuffled to recombine useful mutations in additive or synergistic ways, in effect mimicking the process of natural sexual recombination. Proteins can be ‘bred’ with the appropriate individual properties and then their ‘progeny’ screened for the desired combination of traits. DNA shuffling is a powerful tool enabling rapid and directed evolution of new genes, operons and whole viral genomes.

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References

  • Barnes, W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91:2216–2220; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Bartel, D. P.; Szostak, J. W. Isolation of new ribozymes from a large pool of random sequences. Science 261:1411–1418; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Burbank, L. Short-cuts into the centuries to come: better plants secured by hurrying evolution. In: Whitson, J. W., ed. Luther Burbank, his methods and discoveries: their practical application, Vol. 1. New York: Luther Burbank Press; 1914:176–210.

    Google Scholar 

  • Caldwell, R. C.; Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Meth. Applic. 2:28–33; 1992.

    Google Scholar 

  • Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263:802–805; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Chang, C.-C.J.; Chen, T. T.; Cox, B. W.; Dawes, G. N.; Stemmer, W. P. C.; Punnonen, J.; Patten, P. A. Evolution of a cytokine using DNA family shuffling. Nat. Biotechnol. 17:793–896; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Christians, F. C.; Scapozza, L.; Crameri, A.; Folkers, G.; Stemmer, W. P. C. Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. Nat. Biotechnol. 17:259–264; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Cleland, J. L.; Craik, C. S. Protein engineering: principles and practice. New York: Wiley-Liss; 1996.

    Google Scholar 

  • Crameri, A.; Cwirla, S.; Stemmer, W. P. C. Construction and evolution of antibody-phage libraries by DNA shuffling. Nat. Med. 2:100–102; 1996a.

    Article  PubMed  CAS  Google Scholar 

  • Crameri, A.; Dawes, G.; Rodriguez, E.; Silver, S.; Stemmer, W. P. C. Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat. Biotechnol. 15:436–428; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Crameri, A.; Raillard, S.-A.; Bermudez, E.; Stemmer, W. P. C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288–291; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Crameri, A.; Whitehorn, E. A.; Tate, E.; Stemmer, W. P. C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14:315–319; 1996b.

    Article  PubMed  CAS  Google Scholar 

  • Delagrave, S.; Youvan, D. C. Searching sequence space to engineer proteins: exponential ensemble mutagenesis. Bio/Technol. 11:1548–1552; 1993.

    CAS  Google Scholar 

  • Eigen, M. Steps towards life: a perspective on evolution. Oxford: Oxford University Press; 1993.

    Google Scholar 

  • Gilbert, W. Why genes in pieces? Nature 271:501; 1978.

    Article  PubMed  CAS  Google Scholar 

  • Hellinga, H. W. Rational protein design: combining theory and experiment. Proc. Natl Acad. Sci. USA 94:10015–10017; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Kaufmann, S. A. The origins of order. New York: Oxford University Press; 1993.

    Google Scholar 

  • Kikuchi, M.; Ohnishi, K.; Harayama, S. Novel family shuffling methods for the in vitro evolution of enzymes. Gene 236:159–167; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Leffel, S. M.; Mabon, S. A.; Stewart, C. N., Jr. Applications of green fluorescent protein in plants. BioTechniques 23:912–918; 1997.

    PubMed  CAS  Google Scholar 

  • Liu, D. R.; Magliery, T. J.; Pastrnak, M.; Schultz, P. G. Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. Proc. Natl. Acad. Sci. USA. 94(19):10092–7; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Madigan, M. T.; Marrs, B. L. Extremophiles. Sci. Amer. 276:82–87; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Minshull, J.; Stemmer, W. P. C. Protein evolution by molecular breeding. Curr. Opin. Chem. Biol. 3:284–290; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Moore, J. C.; Arnold, F. H. Directed evolution of a par-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14:458–467; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Ness, J. E.; Welch, M.; Giver, L.; Bueno, M.; Cherry, J. R.; Borchert, T. V.; Stemmer, W. P. C.; Minshull, J. DNA shuffling of subgenomic sequences of substilism. Nat. Biotechnol. 17:893–896; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Ormö, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395; 1996.

    Article  PubMed  Google Scholar 

  • Pang, S.-Z.; DeBoer, D. L.; Wan, Y.; Guanging Ye, J. G. L.; Neher, M. K.; Armstrong, C. L.; Fry, J. E.; Hinchee, M. A. W.; Fromm, M. E. An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol. 112:893–900; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Patthy, L. Modular exchange principles in proteins. Curr. Opin. Struct. Biol. 1:351–361; 1991.

    Article  CAS  Google Scholar 

  • Patthy, L. Exon shuffling and other ways of module exchange. Matrix Biol. 15:301–310; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Patthy, L. Genome evolution and the evolution of exon-shuffling—a review. Gene 238:103–114; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Reihaar-Olson, J.; Bowie, J.; Beyer, R. M.; Hu, J. C.; Knight, K. L.; Lim, W. A.; Mossing, M. C.; Parsell, D. A.; Shoemaker, K. R.; Sauer, R. T. Random mutagenesis of protein sequences using oligonucleotide cassettes. Meth. Enzymol. 208:564–586; 1991.

    Article  Google Scholar 

  • Robertson, D. E.; Mathur, E. J.; Swanson, R. V.; Marrs B. L.; Short, J. M. The discovery of new biocatalysts from microbial diversity. Soc. Ind. Microbiol. News 46:3–8; 1996.

    Google Scholar 

  • Rubingh, D. N. Protein engineering from a bioindustrial point of view. Curr. Opin. Biotechnol. 8:417–422; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Shao, Z.; Arnold, F. H. Engineering new functions and altering existing functions. Curr. Opin. Struct. Biol. 6:513–518; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Stemmer, W. P. C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389–391; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Stemmer, W. P. C. Searching sequence space: using recombination to search more efficiently and thoroughly instead of making bigger combinatorial libraries. Bio/Technol. 13:549–553; 1995.

    Article  CAS  Google Scholar 

  • Stemmer, W. P. C.; Crameri, A.; Ha, K. D.; Brennan, T. M.; Heyneker, J. L. Single-step P.C.R. assembly of a gene and a whole plasmid from large numbers of oligonucleotides. Gene 164:49–53; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Zhang, J.; Dawes, G.; Stemmer, W. P. C. Evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl Acad. Sci. USA 94:4504–4509; 1997.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biological Sciences, University of Calgary, T2N 1N4, Calgary, Alberta, Canada

    Alexandra J. Reid

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  1. Alexandra J. Reid
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Correspondence to Alexandra J. Reid.

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Reid, A.J. DNA shuffling: Modifying the hand that nature dealt. In Vitro Cell.Dev.Biol.-Plant 36, 331–337 (2000). https://doi.org/10.1007/s11627-000-0060-0

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  • DOI: https://doi.org/10.1007/s11627-000-0060-0

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