Using PCR for Rapid Site-Specific Mutagenesis in Large Plasmids

Using polymerase chain reaction (PCR) it is possible to amplify a segment of DNA by a factor of approx 2²⁰ under standard conditions (1 ). Any mutations present in the oligonucleotides used to prime the polymerization reactions will be incorporated in the resulting PCR products. Thus, a specific mutation can be introduced by designing mutagenic PCR primers. This principle has been used to introduce mutations into the DNA of bacterial plasmids (2 –4 ), or fragments of plasmids used for subcloning (5 –10 ). Until very recently, this technique has generally been limited by the size of fragments that can be amplified by a single PCR reaction, which in our hands was about 2700 bp (2.7 kb). When trying to introduce mutations into a plasmid of 3.9 kb, we found that methods for the site-specific mutagenesis by direct amplification of the entire plasmid did not produce amplification products.