Abstract
Chemical pollutants, ionizing radiation, the products of aerobic metabolism and UV light are important metabolic and environmental factors contributing to DNA damage. Ionizing radiation, in particular, induces damage in the form of single-strand breaks, double-strand breaks, crosslinks, and associated conformational changes to the higher-order structure of DNA inside the cell. Several techniques, such as the single cell gel assay are used to analyze strand breakage in subpopulations of cells. Capillary electrophoresis (CE), by virtue of its high-separation efficiency, throughput, and automation has become recognized as a powerful tool to detect base, sequence, and structural changes in nanogram quantities of different DNA substrates. CE can also be combined with mass spectrometry (MS) to detect precursors to DNA strand breaks, i.e., DNA base modifications in human cancer cells (see refs. 1 and 2). In this chapter, we describe methods for the analysis of environment-induced structural changes in linear, plasmid, and genomic DNA. We describe the response of DNA to graded doses of laser or ionizing radiation as examples of how these methods can be used in a practical manner. Using CE to detect small radiation-induced fragments of DNA and conformational changes in large DNA fragments and plasmids provides a faster and more efficient alternative to other techniques such as standard and pulsed-field agarose gel electrophoresis.
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
Le X. C., Xing J. Z., Lee J., Leadon S. A., Weinfeld S. A., and Weinfeld M. (1999) Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage. Science 280, 1066–1069.
Xing J. Z., Carnelley T., Lee J., Watson W. P., Weinfeld M., and Le X. C. (2001) Assay for DNA damage using immunochemical recogition and capillary electrophoresis, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson K. R. and Cheng J., eds.), Humana Press, Totowa, NJ, pp. 419–428.
Siles B. A., Nackerdien Z. E., and Collier G. B. (1997) Analysis of DNA fragmentation using a dynamic size-sieving polymer solution in capillary electrophoresis. J. Chromatogr. A 771, 319–329.
Hammond R. W., Oana H., Schwinefuss J. J., Bonadio J., Levy R. J., and Morris M. D. (1997) Capillary electrophoresis of supercoiled and linear DNA in dilute hydroxy-ethylcellulose solution. Anal. Chem. 69, 1192–1196.
Nevins S. A., Siles B. A., and Nackerdien Z. E. (2000) Analysis of gamma radiation-induced damage to plasmid DNA using dynamic size-sieving capillary electrophoresis. J. Chromatogr. B. 741, 243–255.
Nackerdien Z. E., and Atha D. (1996) Measurement of 60Co-ray induced DNA damage by capillary electrophoresis. J. Chromatogr. B 683, 85–89.
Nackerdien Z., Morris S., Choquette S., Ramos B., and Atha D. (1996) Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis. J. Chromatogr. B 683, 91–96.
Daly M. J., Ouyang L., Fuchs P., and Minton K. (1994) In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans. J. Bacteriol. 176, 3508–3517.
Siles B. A., Anderson D. A., Buchanan N. S., and Warder M. F. (1997) The character-ization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments during capillary electrophoresis. Electrophoresis 18, 1980–1989.
Deforce D. L. and van den Eeckhout E. E. (2001) Analysis of DNA damage using capillary zone electrophoresis and electrospray mass spectrometry, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson K. R. and Cheng J., eds.), Humana Press, Totowa, NJ, pp. 429–441.
Fung E. N. and Yeung E. S. (1995) High-speed DNA-sequencing by using mixed poly(ethylene oxide) solutions in uncoated capillary columns. Anal. Chem. 67, 1913–1919.
Kim Y. and Yeung E. S. (2001) Capillary electrophoresis of DNA fragments using poly(ethylene oxide) as a sieving material, in Capillary Electrophoresis of Nucleic Acids, Vol. 1 (Mitchelson K. R. and Cheng J., eds.), Humana Press, Totowa, NJ, pp. 215–223.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Nackerdien, Z.E., Siles, B.A., Nevins, S.A., Atha, D.H. (2001). Analysis of Environment-Induced DNA Damage by Capillary Electrophoresis. In: Mitchelson, K.R., Cheng, J. (eds) Capillary Electrophoresis of Nucleic Acids. Methods in Molecular Biology, vol 162. Humana Press. https://doi.org/10.1385/1-59259-055-1:407
Download citation
DOI: https://doi.org/10.1385/1-59259-055-1:407
Publisher Name: Humana Press
Print ISBN: 978-0-89603-779-3
Online ISBN: 978-1-59259-055-1
eBook Packages: Springer Protocols