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Separation of Large DNA Molecules by Pulsed-Field Gel Electrophoresis

  • Duncan J. Shaw
Part of the Methods in Molecular Biology™ book series (MIMB, volume 13)

Abstract

Conventional agarose gel electrophoresis is a widely used technique for the analysis of many kinds of biological molecules, including fragments of DNA. It has one major limitation, namely its inability to resolve DNA fragments of greater than approx 30 kb in length. In human genetics, the enormous length of the genome (3 million kb) makes it necessary to have techniques capable of analyzing much larger DNA molecules, and it was in response to this that the methods of pulsed field-gel electrophoresis (PFGE) were developed. The procedure was first described by Schwartz and Cantor (1), and since then numerous variations and modifications have been published (2, 3, 4). These methods all have in common the use of two alternately switched (pulsed) electric fields, arranged at an angle of between 90 and 180 degrees. Conventional gel electrophoresis uses a single, continuous electric field. In PFGE, the molecules are forced to change direction each time the field is switched, and the time taken for a large DNA molecule to reorient itself in response to the change in field is a direct function of its size. Hence, at each pulse, longer molecules become retarded relative to shorter ones because of their longer reorientation time, and over the course of the electrophoretic run, a separation is achieved. The theory of the method was described in detail by Southern et al. (5).

Keywords

Yeast Chromosome Ethidium Bromide Solution Phenyl Methyl Sulfonyl Fluoride Switching Interval Agarose Block 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© The Humana Press, Totowa, NJ 1992

Authors and Affiliations

  • Duncan J. Shaw
    • 1
  1. 1.Department of Medical GeneticsUniversity of Wales College of MedicineCardiffUK

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