PickMutant™ DNA-Shuffling Kit is based on the method Developed by Stemmer. Stemmer introduced DNA shuffling, the first homologous recombination method, in 1994.
DNA shuffling involves the digestion of a gene by DNase I into random fragments, and the reassembly of those fragments into a full-length gene by primerless PCR: the fragments prime on each other based on sequence homology, and recombination occurs when fragments from one copy of a gene anneal to fragments from another copy, causing a template switch, or crossover event. While randomly recombining the DNA sequences, the technique also introduces new point mutations at a relatively high rate (0.7%).
DNA shuffling consists of four steps: (i) preparation of genes to be shuffled, (ii) fragmentation with DNase I, (iii) reassembly by thermocycling in the presence of a DNA polymerase, and (iv) amplification of reassembled products by a conventional PCR.
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