❞ كتاب Directed Evolution Library Creation. Methods and Protocols-Humana Press ❝ ⏤ Frances H. Arnold
Directed evolution has become a powerful tool not only for improving the
utility of enzymes in industrial processes, but also to generate variants that
illuminate the relationship between enzyme sequence, structure, and function. The method most often used to generate variants with random mutations is error-prone PCR. Error-prone PCR protocols are modifications of
standard PCR methods, designed to alter and enhance the natural error rate of
the polymerase (1,2). Taq polymerase (3) is commonly used because of its
naturally high error rate, with errors biased toward AT to GC changes. However, recent protocols include the use of a newly-developed polymerase
whose biases allow for increased variation in mutation type (i.e., more GC to
AT changes) (see Note 1).
Error-prone PCR reactions typically contain higher concentrations of
MgCl2 (7 mM) compared to basic PCR reactions (1.5 mM), in order to stabilize non-complementary pairs (4,5). MnCl2 can also be added to increase the
error-rate (6). Other ways of modifying mutation rate include varying the
ratios of nucleotides in the reaction (7–9), or including a nucleotide analog
such as 8-oxo-dGTP or dITP (10). Fenton et al. (11) describe a mutagenic
PCR protocol that uses dITP as well as provide an analysis of the effects of
dITP and Mn2+ on PCR products. Mutation frequencies from 0.11 to 2% (1 to
20 nucleotides per 1 kb) have been achieved simply by varying the nucleotide ratio and the amount of MnCl2 in the PCR reaction (12). The number of
genes that contain a mutation can also be modified by changing the number
of effective doublings by increasing/decreasing the number of cycles or by
changing the initial template concentration.
Frances H. Arnold
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