Cloning from Gels Following Pulse-Field Gel Electrophoresis

Cloning from DNA fragments fractionated by pulse-field gel electrophoresis (PFGE) offers an opportunity to isolate markers from a specific region of a genome and thus forms part of the armory of the reverse geneticist. In particular, it can be used as an adjunct to chromosome walking and jumping strategies, which are slow procedures if initiated from a single point; isolation of additional start sites facilitates the saturation cloning of a particular region of DNA. The method involves digestion of human genomic DNA with a “rare cutter” endonuclease, fractionation by PFGE, excision of the fragment of interest from the gel, and purification of DNA and its cloning into an appropriate vector (see Fig. 1 for summary).