11] Pulsed-field gel electrophoresis

Abstract

Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single static field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction, with each change of the electric fields. The DNA molecules, thus, migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules, in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE)], separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). A number of models and theories have been proposed to explain some of the more complex behavior of DNA molecules in PFGE. However, biologists rarely need to consult these physical models or equations for practical PFGE applications. This chapter discusses the optimum PFG electrophoretic conditions for the separation of DNA fragments from 1 to 6000 kb.