كتاب Directed Evolution Library Creation. Methods and Protocols-Humana Press

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 George Georgiou - ❰ له مجموعة من الإنجازات والمؤلفات أبرزها ❞ Directed Evolution Library Creation. Methods and Protocols-Humana Press ❝ ❱
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