Mutagenesis gene shuffling

Figure 3.1 Overview of DNA library creation strategies. Random mutagenesis introduces mutations at positions throughout the gene sequence. Semi-rational design randomizes only the specific position(s) of interest. Gene shuffling brings existing sequence diversity from different parental DNA sequences together to form a chimeric library...

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... 

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... 

S. Nirasawa and K. Hayashi. 2008. Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis. Biotechnol. Lett. 30 363-368. 

Figure 2.5 Scheme for DNA shuffling illustrated for the case in which the parent genes originate from the WT by some form of mutagenesis [7e],... 

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). 

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