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)


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).


Yeast Chromosome Ethidium Bromide Solution Phenyl Methyl Sulfonyl Fluoride Switching Interval Agarose Block 
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  1. 1.
    Schwartz, D.C. and Cantor, C. R. (1984) Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75.PubMedCrossRefGoogle Scholar
  2. 2.
    Carle, G. F. and Olson, M. V. (1984) Separahon of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresrs. Nucleic Acids Res. 12, 5647–5664.PubMedCrossRefGoogle Scholar
  3. 3.
    Carle, G. F., Frank, M., and Olson, M. V. (1986) Electrophoretic sepamtions of large DNA molecules by periodic inversion of the electric field. Science 232, 64–68.CrossRefGoogle Scholar
  4. 4.
    Chu, G., Vollrath, D., and Davis, R. W. (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582–1585.PubMedCrossRefGoogle Scholar
  5. 5.
    Southern, E. M., Anand, R., Brown, W. R. A., and Fletcher, D. S. (1987) A model for the separation of large DNA molecules by crossed field gel electrophoresis. Nucleic Acids Res. 15, 5925–5943.PubMedCrossRefGoogle Scholar
  6. 6.
    Snell, R. G. and Wilkins, R.J. (1986) Separation of chromosomal DNA molecules from C. albicans by pulsed field gel electrophoresis. Nucleic Acids Res. 14, 4401–4406.PubMedCrossRefGoogle Scholar
  7. 7.
    Vollrath, D. and Davis, R. W. (1987) Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 15, 7865–7876.PubMedCrossRefGoogle Scholar
  8. 8.
    Van der Ploeg, L. H. T., Schwartz, D. C., Cantor, C. R., and Borst, P. (1984) Antigenic variation in Trypanosoma brucei analysed by electrophoretic separation of chromosome-sized DNA molecules. Cell 37, 77–84.PubMedCrossRefGoogle Scholar
  9. 9.
    Rommens, J. M., Iannuzzi. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L. C., and Collins, F. S. (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245, 1059–1065.PubMedCrossRefGoogle Scholar
  10. 10.
    Bucan, M., Zimmer, M., Whaley, W. L., Poustka, A., Youngman, S., Allitto, B. A., Ormondroyd, E., Smith, B., Pohl, T. M., MacDonald, M., Bates, G. P., Richards, J., Volinia, S., Gilliam, T. C., Sedlacek, Z., Collins, F. S., Wasmuth, J. J., Shaw, D. J., Gusella, J. F., Frischauf, A. M., and Lehrach H. (1990) Physical maps of 4p16. 3, the area expected to contain the Huntington disease mutation. Genomics 6, 1–15.PubMedCrossRefGoogle Scholar
  11. 11.
    Drmanac, R., Petrovic, N., Glisin, V., and Crkvenjakov, R. (1986) A calculation of fragment lengths obtainable from human DNA with 78 restriction enzymes: an aid for cloning and mapping. Nucleic Acids Res. 14, 4691–4692.PubMedCrossRefGoogle Scholar
  12. 12.
    Birren, B. W., Lai, E., Clark, S. M., Hood, L., and Simon, M. I. (1988) Optimised conditions for pulsed field gel electrophoretic separations of DNA. Nucleic Acids Res. 16, 7563–7582.PubMedCrossRefGoogle Scholar

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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