Gene mutagenesis methods directed evolution

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). 

All these techniques create genetic diversity by recombination and point mutations and are well developed. However, insertions and deletions (indels) are also important types of mutation which are probably underrepresented in many conventional mutagenesis strategies. Methods for incorporation of indels in predefined positions in a combinatorial manner have been developed.Although there are some published studies on their use in the directed evolution of biocatalysts,the full potential of these newer methods of gene mutation for enzyme improvement are yet to be demonstrated. 

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... 

The previous sections described structural studies of antibody maturation as a method to understand the evolution of binding (and catalysis) in the immune system. The technique of directed evolution parallels the process of affinity maturation. Both methods use random mutagenesis and gene shuffling, followed by screening and/or selection to identify mutants with the desired function. In contrast to affinity maturation,... 

Whereas site-directed mutagenesis is a technique that focuses on changing very specific residues based on the protein structure, directed evolution and gene shuffling take a more global approach. An advantage of these methods is the limited knowledge of the... 

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