Inverse PCR (Ochman et al., 1988) is a specific application of the general PCR technique. It allows one to introduce mutations into plasmid DNA in a site-specific way, without the need of pre-existing restriction sites. For that purpose, the primers contain the desired mutations in their 5’ ends, together with the recognition sequence of a restriction endonuclease that does not cut the template DNA. The 3’ ends of the primers are complementary to the template DNA region neighbouring the target site of mutagenesis. By DNA amplification with such a primer set, the small fragment between the two primer-binding sites on circular DNA is replaced by the non-complementary sequence of the primers.
The newly introduced restriction site at both ends of amplification products enables the creation of sticky ends for circularization of PCR products. Because the genetic code is degenerate, introduction of this restriction site may occur without altering the local amino acid coding information. Identification of such sites was done by reverse translation of the amino acid sequence region of interest with the computer program SYNCHOIC (Volckaert, 1992). Addition of a hexa-nucleotide at the 5’ end of oligonucleotides was required for functional binding of restriction enzyme onto PCR amplification products. To reduce the background of original plasmid DNA after cloning, template DNA was cleaved between the two primer-binding sites before PCR (only when a suitable restriction site was available).