chapter  49
4 Pages

PFGE: Pulse Field Gel Electrophoresis

Pulse field gel electrophoresis (PFGE) was developed by Carle and Olson in 1985 for the study of yeast Saccharomyces cerevisiae chromosomes. The technique makes it possible to separate DNA fragments of size varying from a few kilobases to over 10,000 kb. The most frequently used size standards are either multimers of the genome of the lambda bacteriophage (from 50 to more than 1000 kb) or chromosomes of Saccharomyces cerevisiae (between 250 and 2500 kb). The DNA fragments are subjected to alternate electric fields in which the relative orientation may vary from 90° to 180°, depending on the systems. The most widely used configuration at present is contour-clamped homogeneous electric fields (CHEF), in which the angle between two electric fields is 120°. The principle of separation is based on the differential speed of reorientation of molecules at the time of alternation of two electric fields. Thus, the large molecules will take more time than small molecules to orient themselves according to the direction of the second electric field. The accumulation of delay at each alternation leads to the separation of the molecules at the end of the migration. Parameters such as agarose percentage, tension applied, or the temperature can be varied to optimize the resolution. However, the principal parameter is the pulsation time, that is, the time of application of an electric field in each direction: this favours the separation of fragments in a given size range.