Abstract
Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to ∼2 lesions/5 Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of nonradioactive DNAs from laboratory, field, or clinical specimens, including higher plants or animals. This method stems from studies of velocity sedimentation of DNA populations, and from the simple methods of agarose gel electrophoresis. Over the last ∼15 years, our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1,2), and electronic imaging for accurate determination of DNA mass. Although all these components improve sensitivity and throughput of large numbers of samples (3,4), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Freeman, S. E., Blackett, A. D., Monteleone, D. C., Setlow, R. B., Sutherland, B. M., and Sutherland, J. C. (1986) Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers. Anal. Biochem. 158, 119–129.
Sutherland, J. C., Reynolds, K. J., and Fisk, D. J. (1996) Dispersion functions and factors that determine resolution for DNA sequencing by gel electrophoresis. Proc. Soc. Photo-Optic. Instrument. Eng. 2680, 326–340.
Sutherland, J. C., Lin, B., Monteleone, D. C., Mugavero, J., Sutherland, B. M., and Trunk, J. (1987) Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA. Anal. Biochem. 163, 446–457.
Sutherland, J. C. (1990) Electronic imaging systems for quantitative electrophoresis of DNA, in Non-invasive Techniques in Biology and Medicine (Freeman, S. E., Fukishima, E., and Green, E. R. eds.) San Francisco Press, San Francisco, CA, pp. 125–134.
Sutherland, B. M., and Shih, A. G. (1983) Quantitation of pyrimidine dimer content of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels. Biochem. 22, 745–749.
Quaite, F. E., Sutherland, B. M., and Sutherland, J. C. (1992) Action spectrum for DNA damage in alfalfa lowers predicted impact of ozone depletion. Nature 358, 576–578.
Quaite, F. E., Sutherland, B. M., and Sutherland, J. C. (1992) Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings. Appl. Theor. Electrophor. 2, 171–175.
Quaite, F. E., Sutherland, J. C., and Sutherland, B. M. (1994) Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation. Plant Mol. Biol. 24, 475–483.
Quaite, F. E., Takayanagi, S., Ruffini, J., Sutherland, J. C., and Sutherland, B. M. (1994) DNA damage levels determine cyclobutyl pyrimidine dimer repair mechanisms in alfalfa seedlings. Plant Cell 6, 1635–1641.
Sutherland, B. M., Quaite, F. E., and Sutherland, J. C. (1994) DNA damage action spectroscopy and DNA repair in intact organisms: alfalfa seedlings, in Stratospheric Ozone Depletion/UV-B Radiation in the Biosphere (Biggs, R. H., and Joyner, M. E. B. eds.) Springer-Verlag, Berlin, pp. 97–106.
Hidema, J., Kumagai, T., Sutherland, J. C., and Sutherland, B. M. (1996) Ultraviolet B-sensitive rice cultivar deficient in cyclobutyl pyrimidine dimer repair. Plant Physiol. 113, 39–44.
Freeman, S. E., Gange, R. W., Matzinger, E. A., and Sutherland, B. M. (1986) Higher pyrimidine dimer yields in skin of normal humans with higher UVB sensitivity. J. Invest. Derm. 86, 34–36.
Freeman, S. E., Gange, R. W., Sutherland, J. C., and Sutherland, B. M. (1987) Pyrimidine dimer formation in human skin. Photochem. PhotoBiol. 46, 207–212.
Freeman, S. E., Gange, R. W., Sutherland, J. C., Matzinger, E. A., and Sutherland, B. M. (1987) Production of pyrimidine dimers in DNA of human skin exposed in situ to UVA radiation. J. Invest. Derm. 88, 430–433.
Freeman, S. E., Hacham, H., Gange, R. W., Maytum, D., Sutherland, J. C., and Sutherland, B. M. (1989) Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ. Proc. Natl. Acad. Sci. USA 86, 5605–5609.
Hacham, H., Freeman, S. E., Gange, R. W., Maytum, D. J., Sutherland, J. C., and Sutherland, B. M. (1990) Does exposure of human skin in situ to 385 or 405 nm UV induce pyrimidine dimers in DNA? Photochem. PhotoBiol. 52, 893–896.
Sutherland, B. M., and Bennett, P. V. (1995) Human white blood cells contain cyclobutyl pyrimidine dimer photolyase. Proc. Natl. Acad. Sci. USA 92, 9732–9736.
Sutherland, J. C., and Sutherland, B. M. (1975) Human photoreactivating enzyme: action spectrum and safelight conditions. Biophys. J. 15, 435–440.
McDonell, M., Simon, M. N., and Studier, F. W. (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110, 119–143.
Doggett, N. A., Smith, C. L., and Cantor, C. R. (1992) The effect of DNA concentration on mobility in pulsed field gel electrophoresis. Nucleic Acids Res. 20, 859–864.
Ribeiro, E., Larcom, L. L., and Miller, D. P. (1989) Quantitative fluorescence of DNA intercalated ethidium bromide on agarose gels. Anal. Biochem. 181, 197–208.
Sutherland, J. C., Monteleone, D. C., Mugavero, J. H., and Trunk, J. (1987) Unidirectional pulsed-field electrophoresis of single-and double-stranded DNA in agarose gels: analytical expression relating mobility and molecular length and their application in the measurement of strand breaks. Anal. Biochem. 162, 511–520.
Southern, E. M. (1979) Measurement of DNA length by gel electrophoresis. Anal. Biochem. 100, 319–323.
Schaffer, H. E., and Sederoff, R. R. (1981) Improved estimation of DNA fragment lengths from agarose gels. Anal. Biochem. 115, 113–122.
Veatch, W., and Okada, S. (1969) Radiation-induced breaks of DNA in cultured mammalian cells. Biophys. J. 9, 330–346.
Bennett, P. V., Gange, R. W., Hacham, H., Hejmadi, V., Moran, M., Ray, S., and Sutherland, B. M. (1996) Isolation of high molecular length DNA from human skin. BioTechniques 21, 458–462.
Bennett, P. V., and Sutherland, B. M. (1993) Quantitative detection of single-copy genes in nanogram samples of human genomic DNA. BioTechniques 15, 520–525.
Chu, G., Vollrath, D., and Davis, R. W. (1986) Separation of large DNA molecules by contour-clamped homogeneous electric field. Science 234, 1582–1585.
Gardiner, K., Laas, W., and Patterson, D. (1986) Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis. Som. Cell Mol. Genet. 12, 185–195.
Serwer, P. (1987) Gel electrophoresis with discontinuous rotation of the gel: An alternative to gel electrophoresis with changing direction of the electrical field. Electrophoresis 8, 301–304.
Sutherland, J. C., Emrick, A. B., and Trunk, J. (1990) Separation of Chromosomal Length DNA Molecules: Pneumatic Apparatus for Rotating Gels During Electrophoresis. Electrophoresis 10, 315–317.
Sutherland, J. C., Sutherland, B. M., Emrick, A., Monteleone, D. C., Ribeiro, E. A., Trunk, J., Son, M., Serwer, P., Poddar, S. K., and Maniloff, J. (1991) Quantitative electronic imaging of gel fluorescence with charged coupled device cameras: applications in molecular biology. BioTechniques 10, 492–497.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Sutherland, B.M., Bennett, P.V., Sutherland, J.C. (1999). DNA Damage Quantitation by Alkaline Gel Electrophoresis. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 113. Humana Press. https://doi.org/10.1385/1-59259-675-4:183
Download citation
DOI: https://doi.org/10.1385/1-59259-675-4:183
Publisher Name: Humana Press
Print ISBN: 978-0-89603-802-8
Online ISBN: 978-1-59259-675-1
eBook Packages: Springer Protocols