UNIT 8.3 Random Mutagenesis by PCR
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Wilson, D. S. and Keefe, A. D. 2001. Random Mutagenesis by PCR. Current Protocols in Molecular Biology. 51:8.3:8.3.1–8.3.9.
- Published Online: 1 MAY 2001
- Published Print: JUL 2000
Error-prone PCR (EP-PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP-PCR, the 5′ and 3′ boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP-PCR doublings performed. The EP-PCR technique described here is for a 400-bp sequence, and an Alternate Protocol is for a library. EP-PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.