Fig. 3. Random mutagenesis techniques. Some of the most widely applied random mutagenesis approaches are represented. (a) Chemical and physical mutagens were the basis of many genome-wide screening experiments, but have not been extensively used in directed evolution. (b) In error-prone PCR, random mutations are introduced by a low fidelity polymerase, resulting in linear DNA fragments with point mutations, which generally require further manipulation to be inserted into an appropriate vector. (c) RCA based methods bypass the need for further manipulation, since the products can be automatically recircularized by the host cells. (d) RAISE was one of the first methodologies designed to introduce random insertions in the sequence to be mutated. Random small extensions are attached to digested fragments of the parental sequence, and full-length genes are reconstructed through PCR. (e) TRINS aims to generate repeats of random short fragments of the parental sequence. First, the parental sequence is digested with DNase I. Part of the obtained fragments are circularized and mixed with the remaining linear fragments. An assembly PCR reaction is then performed. When a linear fragment anneals with a circular fragment, a reaction similar to RCA takes place, leading to the replication of multiple copies of the region corresponding to the circularized fragment. (f) Several mutagenesis techniques based on the mini-Mu transposon have been devised. Such techniques allow the generation of random insertions or even deletions while preserving the appropriate reading frame. (g) Mutator strains were the first tool enabling in vivo random mutagenesis. However, the increased mutagenesis rate applies to the whole genetic material, and not only to the sequence of interest. More sophisticated approaches where only the gene of interest is targeted have been developed. GOI: gene of interest.