Random point mutagenesis

Random point mutagenesis None Exhaustive No multiple simultaneous mutations requires multiple rounds to accumulate beneficial mutations... 

A library of parent DNA sequences encoding for the desired protein is chosen. Sequence diversity is created or increased through a mutagenesis step, either by introduction of random point mutations through error-prone PCR or by recombination of DNA fragments such as DNA shuffling or RACHITT. 

In focused mutagenesis experiments, the challenge is to identify the residues where mutagenesis is likely to be beneficial. Indeed, many successful directed evolution experiments show that mutations occur in regions that would be hard or impossible to predict (and difficull to explain that they do), even when a high-resolution structure and much information about the enzyme is available 14, 98-1001. One possibility is to make use of knowledge gained from early rounds of random point... 

Using a random mutagenesis approach, respiratory-deficient (34) and temperature-sensitive (46, 47) mutants of the Rieske protein of the yeast bc complex have been selected. A large fraction of the point mutants had changes of residues in the bottom of the cluster binding subdomain (the loop /S7-/38) and in the Pro loop comprising residues 174-180 of the ISF (Fig. 9 see Section III,B,3) this indicates the importance of the Pro loop for the stability of the protein. Amino... 

There is some evidence to support the notion that rapid adaptation of function is better achieved by having access to more nonconservative amino-acid substitutions. Still, point mutation methods such as error-prone PCR remain most suitable for attempts to mimic natural evolution or to retrace history. Further functional adaptation may be achieved by saturation mutagenesis at sites identified during random mutagenesis experiments (Miyazaki et al., 1999). 

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