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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Similar content being viewed by others
References
Fischer, S.G., and Lerman, L.S. (1983) Proc. Natl. Acad. Sci. USA 80:1579–1583.
Fischer, S.G., and Lerman, L.S. (1979) Meth. Enzymol. 68:183–191.
Myers, R.M., Lumelsky, N., Lerman, L.S., and Maniatis, T. (1985) Nature 313:495–498.
Myers, R.M., Maniatis, T., and Lerman, L.S. (1986) Meth. Enzymol. 155:501–527.
Myers, R.M., and Maniatis, T. Cold Spring Harbor Symp. Quant. Biol. 51:275-284.
Myers, R.M., Fischer, S.G., Maniatis, T., and Lerman, L.S. (1985) Nucl. Acids Res. 13:3111–3130.
Myers, R.M., Fischer, S.G., Lerman, L.S., and Maniatis, T. (1985) Nucl. Acids Res. 13:3131–3146.
Abrams, E., and Lerman, L.S., personal communication.
Saild, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, HA., and Arnheim, N. (1985) Science 230:1350–1354.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Cold Spring Harbor Symp. Quant. Biol. 51:263–273.
Mullis, K.B., and Faloona, F.A. (1987) Meth. Enzymol. 155:335–350.
Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. (1989) Proc. Natl. Acad. Sci. USA 86:232–236.
Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 239:242–247.
Lerman, L.S., Silverstein, K., and Grinfeld, E. (1986) Cold Spring Harbor Symp. Quant. Biol. 51:285–297.
Lerman, L.S., and Silverstein, K. (1987) Meth. Enzymol. 155:482–501.
Kogan, S.C., Doherty, M., and Gitschier, J. (1987) N. Engl. J. Med. 317:985–990.
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.
Church, G.M., and Kieffer-Higgins, S. (1988) Science 240:185–188.
Gray, M., personal communication; our results, data not shown.
Gyllensten, U., and Erlich, H. (1988) Proc. Natl. Acad. Sci. USA 85:7652–7656.
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.
Pääbo, S., and Wilson, A.C. (1988) Nature 334:387–388.
Dunning, A.M., Talmud, P., and Humphries, S. (1988) Nucl. Acids Res. 16: 10393.
Wong, C., Dowling, C.E., Saiki, R.K., Higuchi, R.G., Erlich, H.A., and Kazazian, H.H. (1987) Nature 330:384–386.
Engelke, D.R., Hoener, P.A., and Collins, F.S. (1988) Proc. Natl. Acad. Sci. USA 85:544–548.
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.
Editor information
Editors and Affiliations
Copyright information
© 1989 Stockton Press
About this chapter
Cite this chapter
Myers, R.M., Sheffield, V.C., Cox, D.R. (1989). Mutation Detection by PCR, GC-Clamps, and Denaturing Gradient Gel Electrophoresis. In: Erlich, H.A. (eds) PCR Technology. Palgrave Macmillan, London. https://doi.org/10.1007/978-1-349-20235-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-349-20235-5_7
Publisher Name: Palgrave Macmillan, London
Print ISBN: 978-0-333-48948-2
Online ISBN: 978-1-349-20235-5
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)