DNA Damage Analysis Using an Automated DNA Sequencer

  • Gopaul Kotturi
  • Wolfgang C. Kusser
  • Barry W. Glickman


Advances in biotechnology and molecular genetics have made possible a better understanding of the molecular nature of mutation. For example, the discovery of genetically altered proto-oncogenes and tumor suppressor genes in cancerous cells has led to a better understanding of the links between mutation and cancer. Similarly, the ability to study mutation and mutational specificity in vivo and in vitro has led to an increased appreciation of the mechanisms of mutation and the role that DNA damage and DNA repair play in determining the specificity of mutagenesis. In turn, differences in both the cellular metabolism of exogenous chemicals and DNA repair can at least in part explain tissue, gender, and species specificity of carcinogenesis. We remain, however, a long way off from being able to predict the individual risks implicated with the mutagenic potential of chemical and physical agents. A part of this problem reflects our lack of knowledge of how individual lesions are handled in different tissues and different species against the genetic makeup of an individual.


Cyclobutane Pyrimidine Dimer Acrylamide Formation Retention Time Shift Salt Front Capillary Electrophoresis Instrument 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ansorge, W., Sproat, B., Stegemann, J., Schwager, C., and Zenke, M. (1987). Automated DNA sequencing: Ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 15:4593–4602.PubMedCrossRefGoogle Scholar
  2. Brash, D., Seetharam, S., Kraemer, K. H., Seidman, M. M., and Bredberg, A. (1987). Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc. Natl. Acad. Sci. USA 84:3782–3786.PubMedCrossRefGoogle Scholar
  3. Chen, D. Y., Swerdlow, H. P., Harke, H. R., Zhang, J. Z., and Dovichi, N. J. (1991). Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis. J. Chromatogr. 559:237–246.PubMedCrossRefGoogle Scholar
  4. Comess, K. M., Burstyn, J. N., Essigmann, J. M., and Lippard, S. J. (1992). Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts. Biochemistry 31:3975–3990.PubMedCrossRefGoogle Scholar
  5. Drobetsky, E. A., and Sage, E. (1993). UV-induced G:C to A:T transitions at the aprt locus of Chinese hamster ovary cells cluster at frequently damaged 5′-TCC-3′ sequences. Mutai. Res. 289:131–136.CrossRefGoogle Scholar
  6. Gao, S., Drouin, R., and Holmquist, G. P. (1994). DNA repair rates mapped along the human PGK1 gene at nucleotide resolution. Science 263:1438–1440.PubMedCrossRefGoogle Scholar
  7. Goodisman, J., and Dabrowiak, J. C. (1992). Quantitative aspects of DNase I footprinting, in:Advances in DNA Sequence Specific Agents (L.H. Hurley, ed.), JAI Press, Greenwich, CT, pp. 25–50.Google Scholar
  8. Gordon, L. K., and Haseltine, W. A. (1982). Quantitation of cyclobutane dimer formation in double and single stranded DNA fragments of defined sequence. Radiat. Res. 89:99–112.PubMedCrossRefGoogle Scholar
  9. Huang, X. C., Quesada, M. A., and Mathies, R. A. (1992). DNA sequencing using capillary array electrophoresis. Anal. Chem. 64:2149–2154.PubMedCrossRefGoogle Scholar
  10. Iwahana, H., Yoshimoto, K., Mizusawa, N., Kudo, E., and Itakura, M. (1994). Multiple fluorescence-based PCR-SSCP analysis. Biotechniques 16:296–305.PubMedGoogle Scholar
  11. Karger, A. E., Harris, J. M., and Gesteland, R. F. (1991). Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis Nucleic Acids Res. 19:4955–4962.PubMedCrossRefGoogle Scholar
  12. Khrapko, K., Hanekamp, J. S., Thilly, W. G., Belenkii, A., Foret, F., and Karger, B. L. (1994). Constant denaturant capillary electrophoresis (CDCE):A high resolution approach to mutational analysis. Nucleic Acids Res. 22:364–369.PubMedCrossRefGoogle Scholar
  13. Koehler, D. R., Awadallah, S. S., and Glickman, B. W. (1991). Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lad correspond with sites of UV-induced mutation in Escherichia coli. J. Biol Chem. 266:11766–11773.PubMedGoogle Scholar
  14. Kunala, S., and Brash, D. E. (1992). Excision repair at individual bases of the Escherichia coli lad gene: Relation to mutation hot spots and transcription coupling activity. Proc. Natl. Acad. Sci. USA 89:11031–11035.PubMedCrossRefGoogle Scholar
  15. Lippke, J. A., Gordon, L. K., Brash, D. E., and Haseltine, W. A. (1981). Distribution of UV light-induced damage in a defined sequence of human DNA:Detection of alkali-sensitive lesions at pyrimidine nucleoside-cytosine. sequences. Proc. Natl. Acad Sci. USA 78:3388–3392.PubMedCrossRefGoogle Scholar
  16. Murov, S. L. (1973). Handbook of Photochemistry, Dekker, New York.Google Scholar
  17. Pfeifer, G. P., Drouin, R., Riggs, A. D., and Holmquist, G. P. (1991). In vivo mapping of a DNA adduct at nucleotide resolution: Detection of pyrimidine (6−4) pyrimidine photoproducts by ligation-mediated polymerase chain reaction. Proc. Natl. Acad. Sci. USA 88:1374–1378.PubMedCrossRefGoogle Scholar
  18. Porcher, C., Malinge, M. C., Picat, C., and Grandchamp, B. (1992). A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automated DNA sequencer. Biotechniques 13:106–113.PubMedGoogle Scholar
  19. Sage, E., Cramb, E., and Glickman, B. W. (1992). The distribution of UV damage in the lad gene of Escherichia coli: Correlation with mutation spectrum. Mutat. Res. 269:285–299.PubMedCrossRefGoogle Scholar
  20. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.PubMedCrossRefGoogle Scholar
  21. Segurado, O. G., and Schendel, D. J. (1993). Identification of predominant T-cell receptor rearrangements by temperature-gradient gel electrophoresis and automated DNA sequencing. Electrophoresis 14:747–752.PubMedCrossRefGoogle Scholar
  22. Shoukry, S., Anderson, M. W., and Glickman, B. W. (1991). A new technique for determining the distribution of N7-methyl guanine using an automated DNA sequencer. Carcinogenesis 12:2089–2092.PubMedCrossRefGoogle Scholar
  23. Shoukry, S., Anderson, M. W., and Glickman, B. W. (1993). Use of fluorescently tagged DNA and an automated DNA sequencer for the comparison of the sequence selectivity of SN1 and SN2 alkylating agents. Carcinogenesis 14:155–157.PubMedCrossRefGoogle Scholar
  24. Smith, L. M., Fung, S., Hunkapiller, M. W., Hunkapiller, T. J., and Hood, L. E. (1985). The synthesis of oligonucleotides containing an aliphatic amino group at the 5′ terminus: Synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 13:2399–2412.PubMedCrossRefGoogle Scholar
  25. Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S.B. H., and Hood, L.E. (1986). Fluorescence detection in automated DNA sequence analysis. Nature 321:674–679.PubMedCrossRefGoogle Scholar
  26. Tornaletti, S., and Pfeifer, G. P., (1994). Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 263:1436–1438.PubMedCrossRefGoogle Scholar
  27. Verpy, E., Biasotto, M., Meo, T., and Tosi, M. (1994). Efficient detection of point mutations on color-coded strands of target DNA. Proc. Natl. Acad. Sci. USA 91:1873–1877.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Gopaul Kotturi
    • 1
  • Wolfgang C. Kusser
    • 1
  • Barry W. Glickman
    • 1
  1. 1.Centre for Environmental Health, Department of BiologyUniversity of VictoriaVictoriaCanada

Personalised recommendations