title: Random mutagenesis by PCR.
abstract: 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.
authors.affiliation: Zyomyx, Hayward, California, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Wilson
authors.forename: A D
authors.initials: AD
authors.lastname: Keefe
mesh_terms: D005821:Genetic Techniques; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D019914:Taq Polymerase