Abstract
Denaturing gradient gel electrophoresis (DGGE) is a robust method for point mutation detection that has been widely used for many years (1). It is a polymerase chain reaction (PCR)-based method, the principle being the altered denaturing temperature of a PCR product with a mutation compared to the wild-type product (see Chapter 6). PCR performed on DNA of an individual with a point mutation in one of two genes will lead to a mixture of different products. PCR products from both the wild-type gene and the mutated gene will be formed. These are known as the homoduplex products. The difference in melting temperature between these two products, however, is subtle. Another type of product, heteroduplexes, consisting of a wildtype strand combined with a mutant strand of DNA, will also be formed during the last cycles of the reaction. The real strength of DGGE lies in the fact that the heteroduplex PCR products will have much lower melting temperatures compared to the homoduplex PCR products, because the heteroduplexes have a mismatch (see Fig. 1).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sheffield, V. C., Cox, D. R., Lerman, L. S., and Meyers, 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.
Wu, Y., Hayes, V. M., Osinga, J., et al. (1998) Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Nucleic Acids Res. 26, 5432–5440.
Krawczak, M., Reiss, J., and Cooper, D. N. (1992) The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum. Genet. 90, 41–54.
van der Luijt, R. B., Khan, P. M., Vasen, H. F., et al. (1997) Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis. Hum. Mutat. 9, 7–16.
Wagner, A., Hendriks, Y., Meijers-Heijboer, E. J., et al. (2001) Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree. J. Med. Genet. 38, 318–322.
Wijnen, J., Vasen, H., Khan, P. M., et al. (1995) Seven new mutations in hMSH2, an HNPCC gene, identified by denaturing gradient-gel electrophoresis. Am. J. Hum. Genet. 56, 1060–1066.
den Dunnen, J. T., Grootscholten, P. M., Bakker, E., et al. (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet. 45, 835–847.
Hayes, V. M. (2003) Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay. Hum. Mutat. 22, 59–66.
Kerem, B., Rommens, J. M., Buchanan, J. A., et al. (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073–1080.
Soussi, T. and Beroud, C. (2001) Assessing TP53 status in human tumours to evaluate clinical outcome. Nature Rev. Cancer 1, 233–240.
Nedergaard, T., Guldberg, P., Ralfkiaer, E., and Zeuthen, J. (1997) A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin’s lymphoma. Int. J. Cancer 71, 364–369.
Hartmann, A., Blaszyk, H., McGovern, R. M., et al. (1995) p53 gene mutations inside and outside of exons 5-8: the patterns differ in breast and other cancers. Oncogene 10, 681–688.
Tannock, G. W. (2002) Analysis of the intestinal microflora using molecular methods. Eur. J. Clin. Nutr. 56(Suppl. 4), S44–S49.
Favier, C. F., Vaughan, E. E., De Vos, W. M., and Akkermans, A. D. (2002) Molecular monitoring of succession of bacterial communities in human neonates. Appl. Environ. Microbiol. 68, 219–226.
Donskey, C. J., Hujer, A. M., Das, S. M., et al. (2003) Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients. J. Microbiol. Methods 54, 249–256.
Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Roelfsema, J.H., Peters, D.J.M. (2005). Denaturing Gradient Gel Electrophoresis (DGGE). In: Walker, J.M., Rapley, R. (eds) Medical Biomethods Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-870-6:079
Download citation
DOI: https://doi.org/10.1385/1-59259-870-6:079
Publisher Name: Humana Press
Print ISBN: 978-1-58829-288-9
Online ISBN: 978-1-59259-870-0
eBook Packages: Springer Protocols