Random mutagenesis by PCR

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....

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Bibliographic Details
Published inCurrent protocols in molecular biology (Print) Vol. Chapter 8; p. Unit8.3
Main Authors Wilson, D S, Keefe, A D
Format Journal Article
LanguageEnglish
Published United States 01.05.2001
Subjects
Genetic Techniques
Mutagenesis
Point Mutation
Polymerase Chain Reaction - methods
Taq Polymerase - metabolism
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Summary: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.
ISSN:1934-3647
DOI:10.1002/0471142727.mb0803s51