Pulsed-Field Gel Electrophoresis

DNA undergoing electrophoresis in agarose assumes a conformation that only permits the movement of molecules up to about 20 kb in size. Beyond this limit, mobility rapidly decreases as the molecules become trapped in the agarose matrix. A reduction in agarose concentration to 0.5% and the application of a reduced voltage permits the resolution to be increased to a least 50 kb (1 ), but gels of this concentration and less are too fragile and difficult to manipulate and run times become excessively long. Since the introduction of pulsed-field gel electrophoresis (PFGE) in 1983 (2 ), this technique has been used to separate molecules as large as 12 Mb (3 ). The technique relies on a direct current (D.C.) electric field that periodically changes direction and/or intensity relative to the agarose gel. The time interval during which the field is in any one direction is called the pulse time, and its duration is the single most important factor in determining the molecular size range over which separation is possible. In simplistic terms, large DNA molecules are able to migrate through the agarose matrix by zigzagging in response to changes in the electric field and this process has been confirmed by direct visualization (4 –6 ). Short pulse times, resulting in rapid changes in field direction, allow smaller molecules to migrate, whereas larger molecules cannot respond quickly enough and become trapped in the matrix. As the pulse time is increased, progressively larger molecules are able to migrate, but the resolution of smaller molecules is decreased. The size window of separation is influenced primarily by the choice of pulse time.