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Nucleic Acids Research, 1990, Vol. 18, No. 17 5107-5112
© 1990

MOLECULAR BIOLOGY

Solid phase in vitro mutagenesis using plasmid DNA template

Thomas Hultman, Maria Murby, Stefan Stähl, Erik Hornes1 and Mathias Uhlén*

Department of Biochemistry and Biotechnology, Royal Institute of Technology S-100 44 Stockholm, Sweden 1Apothekarnes Laboratorium A.S. Harbitzalléen 3, 0275 Oslo 2, Norway

*To whom correspondence should be addressed

Received June 7, 1990. Revised July 23, 1990. Accepted July 23, 1990.

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coil host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.


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