Fig. 4. Recombination-based mutagenesis techniques. A set of some of the main mutagenesis techniques based on recombination is displayed. (a) DNA shuffling was the first recombination-based in vitro mutagenesis technique to be developed. A set of homologous sequences is treated with DNase I, and the resulting mix of fragments is used to reassemble full-length sequences through self-priming PCR. (b) In StEP, a set of homologous sequences is used as templates for a series of cycles of annealing with primers and extension of the primers by a DNA polymerase. In each cycle, the growing primers can anneal with a different template, resulting in chimeric full-length sequences. (c) RACHITT requires less homology between parental sequences than DNA shuffling and StEP. A set of fragments obtained by treatment with DNase I of the complementary strands of the sequences to be recombined is hybridized to a single-stranded copy of one of the parental sequences. After digesting overhangs, filling the gaps and ligating the nicks, the scaffold strands are digested, and double-stranded chimeric sequences are obtained by means of PCR. (d) ITCHY decreases even further the sequence homology requirements, but it is limited to a single crossover per variant. Exonuclease III is used to incrementally truncate one of the parental genes from its 3′ end, and the other one from its 5′ end. Then, random-length fragments of each gene are ligated. GOI: gene of interest.