Abstract
DNA shuffling is a developed technique that allows accelerated and directed protein evolution in vitro. In this method, the acquisition of genes encoding improved proteins is done in two steps: First, a single gene is mutagenized, and desired mutant genes are selected; second, the mutant genes are fragmented by DNase I, and subsequently recombined in vitro, by using polymerase chain reaction (PCR). Among the recombinants (i.e., the products of DNA shuffling), those producing most favored proteins are isolated (1,2) Fig. 1). Modified versions of the DNA shuffling exist: Random priming was used to generate DNA fragment, instead of the DNase I digestion (3) Fig. 1); PCR conditions, with very short annealing/extension steps, were employed to increase the frequency of recombination (4). Using these techniques, a number of improved enzymes have been obtained (1–9). When the DNA shuffling is done using a set of homologous genes, instead of a set of mutant genes derived from a single gene, this technique is called “family shuffling” Fig. 2). Family shuffling utilizes naturally occurring nucleotide substitutions among family genes as the driving force for the in vitro evolution. The application of the family shuffling strategy has also provided many successful examples (10–15).
Keywords
- Polymerase Chain Reaction
- Polymerase Chain Reaction Product
- Helper Phage
- Shuffling Product
- Deoxythymidine Triphosphate
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.
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Kikuchi, M., Harayama, S. (2002). DNA Shuffling and Family Shuffling for In Vitro Gene Evolution. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology™, vol 182. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-194-9:243
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DOI: https://doi.org/10.1385/1-59259-194-9:243
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