Protocol

Directed Evolution Library Creation

Volume 231 of the series Methods in Molecular Biology™ pp 3-9

Generating Mutant Libraries Using Error-Prone PCR

  • Patrick C. CirinoAffiliated withDivision of Chemistry and Chemical Engineering, California Institute of Technology
  • , Kimberly M. MayerAffiliated withBiology Department, Brookhaven National Laboratory
  • , Daisuke UmenoAffiliated withDivision of Chemistry and Chemical Engineering, California Institute of Technology

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Abstract

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).