Mutation Detection by PCR, GC-Clamps, and Denaturing Gradient Gel Electrophoresis

  • Richard M. Myers
  • Val C. Sheffield
  • David R. Cox

Abstract

Denaturing gradient gel electrophoresis (DGGE) allows the separation of DNA molecules differing by as little as a single base change.1-5 The separation is based on the melting properties of DNA in solution. DNA molecules melt in discrete segments, called melting domains, when the *temperature or denaturant concentration is raised. Melting domains vary from about 25 base pairs (bp) to several hundred bp in length, and each melts cooperatively at a distinct temperature called a Tm. Due to the considerable contribution of stacking interactions between adjacent bases on a DNA strand to double helical stability, the Tm of a melting domain is highly dependent on its nucleotide sequence. The Tms of DNA fragments differing by even very small changes, such as a single base substitution, can differ by as much as 1.5°C. In the DGGE system, DNA fragments are electrophoresed through a polyacrylamide gel that contains a linear gradient, from top to bottom, of increasing DNA denaturant concentration. As a DNA fragment enters the concentration of denaturant where its lowest temperature melting domain melts (equivalent to the Tm of the domain), the molecule forms a branched structure that has a retarded mobility in the gel matrix. If the gradient conditions are chosen properly, DNA fragments differing by single base changes begin branching, and hence slowing down, at different positions in the gel, resulting in the separation of the fragments at the end of the electrophoretic run.

Keywords

Migration Phenol EDTA Bromide Electrophoresis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Fischer, S.G., and Lerman, L.S. (1983) Proc. Natl. Acad. Sci. USA 80:1579–1583.CrossRefGoogle Scholar
  2. 2.
    Fischer, S.G., and Lerman, L.S. (1979) Meth. Enzymol. 68:183–191.CrossRefGoogle Scholar
  3. 3.
    Myers, R.M., Lumelsky, N., Lerman, L.S., and Maniatis, T. (1985) Nature 313:495–498.CrossRefGoogle Scholar
  4. 4.
    Myers, R.M., Maniatis, T., and Lerman, L.S. (1986) Meth. Enzymol. 155:501–527.CrossRefGoogle Scholar
  5. 5.
    Myers, R.M., and Maniatis, T. Cold Spring Harbor Symp. Quant. Biol. 51:275-284.Google Scholar
  6. 6.
    Myers, R.M., Fischer, S.G., Maniatis, T., and Lerman, L.S. (1985) Nucl. Acids Res. 13:3111–3130.CrossRefGoogle Scholar
  7. 7.
    Myers, R.M., Fischer, S.G., Lerman, L.S., and Maniatis, T. (1985) Nucl. Acids Res. 13:3131–3146.CrossRefGoogle Scholar
  8. 8.
    Abrams, E., and Lerman, L.S., personal communication.Google Scholar
  9. 9.
    Saild, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, HA., and Arnheim, N. (1985) Science 230:1350–1354.CrossRefGoogle Scholar
  10. 10.
    Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Cold Spring Harbor Symp. Quant. Biol. 51:263–273.CrossRefGoogle Scholar
  11. 11.
    Mullis, K.B., and Faloona, F.A. (1987) Meth. Enzymol. 155:335–350.CrossRefGoogle Scholar
  12. 12.
    Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. (1989) Proc. Natl. Acad. Sci. USA 86:232–236.CrossRefGoogle Scholar
  13. 13.
    Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 239:242–247.CrossRefGoogle Scholar
  14. 14.
    Lerman, L.S., Silverstein, K., and Grinfeld, E. (1986) Cold Spring Harbor Symp. Quant. Biol. 51:285–297.CrossRefGoogle Scholar
  15. 15.
    Lerman, L.S., and Silverstein, K. (1987) Meth. Enzymol. 155:482–501.CrossRefGoogle Scholar
  16. 16.
    Kogan, S.C., Doherty, M., and Gitschier, J. (1987) N. Engl. J. Med. 317:985–990.CrossRefGoogle Scholar
  17. 17.
    Myers, R.M., Sheffield, V., and Cox, D.R. (1988) in Genomic Analysis: A Practical Approach. K. Davies, ed. IRL Press Limited, Oxford, pp. 95–139.Google Scholar
  18. 18.
    Church, G.M., and Kieffer-Higgins, S. (1988) Science 240:185–188.CrossRefGoogle Scholar
  19. 19.
    Gray, M., personal communication; our results, data not shown.Google Scholar
  20. 20.
    Gyllensten, U., and Erlich, H. (1988) Proc. Natl. Acad. Sci. USA 85:7652–7656.CrossRefGoogle Scholar
  21. 21.
    Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, SJ., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239:487–491.CrossRefGoogle Scholar
  22. 22.
    Pääbo, S., and Wilson, A.C. (1988) Nature 334:387–388.CrossRefGoogle Scholar
  23. 23.
    Dunning, A.M., Talmud, P., and Humphries, S. (1988) Nucl. Acids Res. 16: 10393.CrossRefGoogle Scholar
  24. 24.
    Wong, C., Dowling, C.E., Saiki, R.K., Higuchi, R.G., Erlich, H.A., and Kazazian, H.H. (1987) Nature 330:384–386.CrossRefGoogle Scholar
  25. 25.
    Engelke, D.R., Hoener, P.A., and Collins, F.S. (1988) Proc. Natl. Acad. Sci. USA 85:544–548.CrossRefGoogle Scholar
  26. 26.
    Myers, R.M., Sheffield, V.C., and Cox, D.R. (1989) in The Polymerase Chain Reaction. R. Gibbs, H. Kazazian, and H. Erlich, eds.; Cold Spring Harbor Press, Cold Spring Harbor, NY, in press.Google Scholar

Copyright information

© Stockton Press 1989

Authors and Affiliations

  • Richard M. Myers
  • Val C. Sheffield
  • David R. Cox

There are no affiliations available

Personalised recommendations