Abstract
Denaturing-gradient gel electrophoresis (DGGE) detects DNA sequence differences. Thus, it can be used to screen for point mutations or other types of mutation prior to DNA sequence analysis. The technique, first described by Fischer and Lerman (1), entails electrophoresis of DNA fragments at high temperature (approx. 60°C) in an acrylamide gel that contains a gradient of denaturant (formamide and urea). As a DNA fragment migrates in the gel, it encounters increasing concentrations of denaturant and at some point, it will become partially or totally single-stranded (melted, denatured). The position at which the DNA melts is determined by its nucleotide sequence and composition. Partial or complete denaturation causes a marked decrease in the electrophoretic mobility of the DNA and results in bands that are usually quite sharp. Two DNA fragments of the same size but of different sequence frequently will denature at different points within the gradient, and will therefore be separable by DGGE. In general, A-T-rich sequences denature at lower denaturant concentrations than G-C rich sequences. The method is applicable for fragments that are about 50–1000 bp long (2,3).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fischer, S. G. and Lerman, L. S. (1979) Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell 16, 191–200.
Myers, R. M., Maniatis, T., and Lerman, L. S. (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 155, 501–527.
Lerman, L. S. and Silverstein, K. (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol. 155, 482–501.
Lerman, L. S., Silverstein, K., and Grinfeld, E. (1986) Searching for gene defects by denaturing gradient gel electrophoresis. Cold Spring Harbor Symp. Quant. Biol. 51, 285–297.
Fischer, S. G. and Lerman, L. S. (1983) DNA fragments differing by single-base pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579–1583.
Myers, R. M., Fischer, S. G., Lerman, L. S., and Maniatis, T. (1985) Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13, 3131–3146.
Dlouhy, S. R., Schaff, D. A., Trofatter, J. A., Liu, H. S., Stambrook, P. J., and Tischfield, J. A. (1989) Denaturing gradient gel analysis of single-base substitutions at a mouse adenine phosphoribosyltransferase splice acceptor site. Mol. Carcinogenesis 2, 217–225.
Myers, R. M., Lumelsky, N., Lerman, L. S., and Maniatis, T. (1985) Detection of single base substitutions in total genomic DNA. Nature 313, 495–498.
Noll, W. W. and Collins, M. (1987) Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique. Proc. Natl. Acad. Sci. USA 84, 3339–3343.
Cariello, N. F., Scott, J. K., Kat, A. G., Thilly, W. G., and Keohavong, P. (1988) Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRTMunich. Am. J. Hum. Genet. 42, 726–734.
Amselem, S., Duquesnoy, B. S., Attree, O., Novelli, G., Bousnina, S., Postel-Vinay, M-C., and Goossens, M. (1989) Laron dwarfism and mutations of the growth hormone-receptor gene. N. Engl. J. Med. 321, 989–995.
Theophilus, B. D. M., Latha, T., Grabowsky, G. A., and Smith, F. I. (1989) Comparison of RNase A, a chemical cleavage and GC-clamped denaturing gradient gel electrophoresis for the detection of mutations in exon 9 of the human acid β-glucosidase gene. Nucleic Acids Res. 17, 7707–7722.
Sheffield, V. C., Cox, D. R., Lerman, L. S., and Myers, R. M. (1989) Attachment of a 40-base-pair G + C rich sequence (GC clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. USA 86, 232–236.
Myers, R. M., Fischer, S. G., Maniatis, T., and Lerman, L. S. (1985) Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13, 3111–3129.
Keohavong, P. and Thilly, W. (1989) Fidelity of DNA polymerases in DNA amplification. Proc. Natl. Acad. Sci. USA 86, 9253–9275.
Myers, R. M., Sheffield, V. C., and Cox, D. R. (1989) Mutation detection by PCR, GC-clamps, and denaturing gradient gel electrophoresis, in PCR Technology (Erlich, H. A., ed.) Stockton, NY, pp. 71–88.
Maniatis, T., Fritsch, E. F., and Sambrook, J. (eds.) (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 173–177.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Dlouhy, S.R., Wheeler, P., Trofatter, J.A., Stambrook, P.J., Tischfield, J.A. (2000). Detecting Point Mutations by Denaturing-Gradient Gel Electrophoresis. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:705
Download citation
DOI: https://doi.org/10.1385/1-59259-038-1:705
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
eBook Packages: Springer Book Archive