PFGE is an extension of the standard agarose gel electrophoresis of restriction enzyme fragmented DNA. The main difference is that PFGE can be applied to very large DNA molecules matching the size of a complete bacterial genome (3-4 million base pairs) and that it provides size-dependent resolution of large restriction fragments (up to hundreds of thousands base pairs). The approach is based on the introduction of alternating voltage gradient to the large DNA fragments. The steps in the PFGE procedure include a gentle lysis of the microbial cells immobilized in an agarose block. This is followed by digestion with a restriction enzyme belonging to the "rare cutters", which have only a small number of restriction sites in the bacterial genome. If the whole genome sequence of the strain of interest is known, this can be utilized in the selection of the restriction enzyme. After digestion, the bacterial DNA fragments, still in the agarose block, are subjected to PFGE using settings, which depend on the expected fragment size distribution. After staining, the gel is visualized and photographed. The banding pattern is compared to that of the strain of interest.
The PFGE method is currently undergoing standardization by the European Committee on Standardization (CEN). Biosafe Ltd has been contracted as the project lead to prepare the standard.