Part of the Methods in Molecular Biology™ book series (MIMB, volume 231)
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.
KeywordsEDTA DMSO Manganese Recombination Agarose
- 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
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