Gene mutagenesis methods

To date, an impressive number of gene mutagenesis methods are available for application in directed evolution 17-20,36,37). However, it is currently not clear how they compare in terms of efficiency and ease of performance. It is also not obvious when and how to apply a given method in a directed evolution project 36-38). The fact that a few of the methods constitute proprietary intellectual property, such as DNA shuffling, poses a different kind of problem for potential users in industry. Some of the most important gene mutagenesis methods are described briefly here (for complete coverage, the reader is referred to recent reviews 17-20,36,37). 

Directed evolution involves the proper combination of molecular biological methods for random gene mutagenesis and gene expression [10], coupled with appropriate high-throughput screening systems [11], which allow rapid determination of the enantiomeric purity of a chiral product. Typically, thousands of samples... 

In addition to saturation mutagenesis and related protocols, shuffling-based methods have also benefited from easy and reliable synthesis of DNA. The original gene-shuffling method that hinges upon random fragmentation followed by PCR... 

Fig. 3 The comparison between random mutagenesis methods and gene recombination methods. Random mutagenesis methods create a library of variants containing point mutations or insertions/deletions (represented by x) from a single parental gene, whereas gene recombination methods create a library of chimeric variants via blockwise exchange of sequence information among the parental genes. A few representative methods that have been developed so far are listed.

Horton, R. (1993) In vitro recombination and mutagenesis of DNA SOEing together tailor-made genes, in Methods in Molecular Biology, vol. 15 PCR Protocols Current Methods and Applications (White, B. A., ed.), Humana, Totowa, NJ, pp. 251-261. 

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