The Introduction of PFGE

The concept of subjecting chromosomal DNA of microorganisms to two alternating electric fields for separation of large DNA fragments (40 to 2000 kb) within agarose gels was introduced in 1984 by Schwartz and Cantor. Subsequently, a variety of alternative electrophoretic configurations, using currents "pulsed" in different directions over controlled time intervals, have been developed. These include orthogonal field alternation gel electrophoresis (Carle and Olson, 1984), vertical alternating field gradient gel electrophoresis (Gardiner et al., 1986), periodic field inversion gel electrophoresis (Carle et al., 1986), and contour-clamped homogeneous electric field electrophoresis (CHEF) (Chu et al., 1986).

CHEF coupled with a programmable autonomously controlled electrode gel electrophoresis (PACE) have become the most common pulsed field methods used for DNA fingerprinting (Clark et al., 1988). Both systems contain three major components: a power module to generate the electrode voltages and to store switching function parameters, a cooling module to keep the temperature at 14°C, and an electrophoresis chamber. The chamber contains 24 horizontal electrodes, some of which are clamped to eliminate DNA lane distortion. The electrodes are arranged in a hexagon that offers reorientation angles of 60 or 120 degrees, in contrast to traditional orthogonal field alternation gel systems with two perpendicular electrodes. The resolution of PFGE is dramatically affected by the number and configuration of the electrodes used, because these alter the shape of the applied electrical field. For high-resolution separation, the most effective electrode configurations yield angles of more than 110 degrees (Cantor et al., 1988). In PACE, each electrode's voltage is independently controlled and can generate an unlimited number of electric fields of different voltage gradients, orientations, and intervals sequentially in time, whereas the traditional CHEF systems are limited to two alternating electric fields at a fixed reorientation angle. Both CHEF and PACE technologies are best configured to offer a unique tool to distinguish large-molecular-weight DNA molecules.

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