Pulsed Field Gel Electrophoresis

How Does PFGE Work?

PFGE stemmed from the observation that DNA molecules elongate upon application of an electric field and return to an unelongated state upon removal of the electric field; this relaxation rate is dependent on the size of the DNA.

When the orientation of the electric field is changed during electrophoresis, the DNA molecules must return to their elongated form prior to reorientation, thus affecting the migration rate. This effect can be used to greatly extend the size range over which electrophoretic DNA separations are possible.

When the electrical field is applied to the gel, the DNA molecules elongate in the direction of the electrical field. The first electrical field is then switched to the second field according to the run specifications. The DNA must change conformation and reorient before it can migrate in the direction of this field.

As long as the alternating fields are equal with respect to the voltage and pulse duration, the DNA will migrate in a straight path down the gel (see below).

Time-lapse representation of DNA molecules undergoing PFGE.

Considerations For PFGE Sample Preparation

The large size of DNA molecules to be separated by PFGE imposes certain constraints on sample preparation and handling. High molecular weight DNA is easily cleaved through shearing and imparts very high solution viscosity. For these reasons, DNA samples for PFGE are generally prepared by embedding in gel medium. Cellular source material is suspended in low gelling agarose and the gelled suspension is poured into molds. All subsequent manipulations (for example, cell lysis, protein removal, and restriction digestion) are performed by diffusing reagents into the resultant gel plugs. The processed gel plugs are then carefully loaded into wells of an agarose gel used for PFGE.

Find more information on troubleshooting PFGE gels, plug preparation, preparing and running a gel, image analysis, PFGE size standards, restriction enzyme digestion, and critical parameters in optimizing sample resolution in the Protocols section below.

Different Types of Pulsed Field Gel Electrophoresis

• CHEF (clamped homogeneous electrical field) technology resolves DNA over a wide range of molecular weights in a straight lane; it employs the principles of contour-clamped electrophoresis to generate homogenous electrical fields. More information on CHEF
• PACE (programmable autonomously controlled electrodes) technology allows users to select the angle of electrophoretic pulsing optimal for the desired size range
• DR (dynamic regulation) is the electronics design by which each of the 24 electrodes is regulated; CHEF-DR^® systems are capable of compensating for changes in buffer conductivity or gel size, preventing these changes from affecting the reproducibility of results
• FIGE (field inversion gel electrophoresis) is used for rapid sample resolution in the 100 bp to 250 kb size range; in FIGE the electrical field is fixed at 1 angle (180°) and is inverted in the forward and reverse directions
• AFIGE (asymmetric field inversion gel electrophoresis) is a further refinement of the FIGE technology; AFIGE applies a different voltage to the forward direction electrical field than to the reverse direction electrical field, which optimizes the sample resolution in the FIGE size range

References

Klotz LC and Zimm BH (1972). Retardation times of deoxyribonucleic acid solutions. II. Improvements in apparatus and theory. Macromolecules 5, 471–481.

Schwartz DC and Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75.