A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.
A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.
Urban
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Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität, Universitätsstrasse 150, D-44780 Bochum, Germany.
Neukirchen
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Jaeger
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D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008049:Lipase / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D037401:Scyphozoa
D004269:DNA, Bacterial; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008049:Lipase; C074326:CTTAAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases