Isolation of plant DNA for pulsed-field gel electrophoresis

A prerequisite for physical mapping by pulsed-field gel electrophoresis (PFGE) is the saturation of a given genome or chromosomal region with single copy markers. One possible way to achieve this goal is by construction of a saturated restriction fragment length polymorphism (RFLP) map. Although RFLP maps are now available for many plant species, only a few systems provide the high density of markers (at least one marker every 1000 kb) required for long-range physical mapping using PFGE. At present, only four plant systems, Arubidofm (1,2), tomato (3), potato (4,5), and rice (6), have a sufftcient density of markers. These species are characterized by relatively small genomes when compared to other plants, with an average distance between individual RFLP markers of 400-800 kb. PFGE in combination with the digestion of DNA, using rare-cutting restriction enzymes, is able to bridge these gaps, and will allow the construction of long-range physical maps for regions of these plant genomes (7). PFGE has already been used in a number of species to construct long-range restriction maps of a number of gene families and repeated DNA sequences (8-12).