Generating Mutant Libraries Using Error-Prone PCR
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) (seeNote 1).
- 1.Leung, D. W., Chen, E., and Goeddel, D. V. (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11–15.Google Scholar
- 8.Nishida, Y. and Imanaka, T. (1994) Alternation of substrate specificity and optimum pH of sarcosine oxidase by random and site-directed mutagenesis. Appl. Environ. Microbiol. 60, 4213–4215.Google Scholar
- 12.Zhao, H., Moore, J. C., Volkov, A. A., and Arnold, F. H. (1999) Methods for optimizing industrial enzymes by directed evolution, in Manual of Industrial Microbiology and Biotechnology ASM, Washington, DC, pp. 597–604.Google Scholar
- 13.Clontech, Palo Alto, CA (1999) Diversify™ PCR Random Mutagenesis Kit. CLONTECHniques 14, 14–15.Google Scholar