Solid phase in vitro mutagenesis using plasmid DNA template.
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 coli 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.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
T
T
Hultman
M
M
Murby
S
S
Ståhl
E
E
Hornes
M
M
Uhlén
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D014169:Transformation, Bacterial