Pulsed-Field Gel Electrophoresis of Bacterial DNA

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Abstract
The limit of fragment size of DNA separated by conventional agarose electrophoresis (approximately 50 kb) can be increased by introducing a pulse or change of direction to the electric field. Before the early 1990s, the mechanism by which separation of large DNA molecules in pulsed-field gel electro-phoresis (PFGE) was achieved was not fully understood, al-though many theories existed. Time-lapse photomicroscopy of DNA molecules in an agarose gel under pulsed electric fields then showed the path of the molecules. Under the influence of the first electric field the DNA elongates in the direction of the field, but when the field is switched to a different orientation many points along the DNA molecule are pulled by the field until one end eventually leads the molecule in the new direction. When the first field is reapplied, the same reorientation process occurs. The speed of reorientation is dependent on and proportional to the DNA fragment size, and it is this reorientation that provides the parameter by which the fragments are separated.

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