Mutagenesis methods

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

Systematic experimental and theoretical studies of this kind are helpful in performing in vitro evolution of enantioselectivity. Nevertheless, several questions are not fully answered. Are remote mutations more important than those close to the active site, or is the opposite true Is it more effective to allow randomization all over the enzyme rather than focusing on the region around the active site (or vice versa) To be sure, when applying epPCR or any other mutagenesis method that more or less... 

Seng Wong, T., Roccatano, D., Loakes, D. et al. (2007) Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+) a random mutagenesis method with consecutive nucleotide exchanges that complements the bias... 

The design of any DCC experiment involves several components, loosely aligned with the components of a system undergoing Darwinian evolution (Fig. 1.4) (1) a library of building blocks (components of a population), (2) a reversible reaction (analogous to a mutagenesis method or reproduction), (3) a selection mechanism, and (4) an analytical method. The relatively short history of DCC has seen many innovative approaches to... 

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). 

In 1989, a very practical mutagenesis method was described by Leung et al. 39), which was later improved by Cadwell and Joyce 40). Error-prone polymerase chain reaction (epPCR), as it is called, is based on the classical DNA amplification... 

In general, the published mutagenesis methods fall into two categories (Kurtzman,... 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

The improvement in enantioselectivity by the directed evolution of Pseudomonas aeruginosa lipase is shown in Figure 8(b).8 The combination of different mutagenesis methods (error-prone PCR and site-specific saturation mutagenesis) improved the enantioselectivity from E=l.l in wild type to E=25.8. 

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