DNA Sequencing by Chemiluminescent Detection

  • Stephan Beck
Part of the Methods in Molecular Biology™ book series (MIMB, volume 23)


The development of fluorescent, calorimetric, and more recently chemiluminescent detection systems have opened the way to replace radioactive detection for DNA sequencing (1–3). Although the bulk of DNA sequencing is still carried out using radioactive detection, the availability of better chemistries, equipment, and protocols make nonradioactive detected DNA sequencing increasingly popular. This chapter describes a protocol for standard Sanger M 13 DNA sequencing (4,5) using enzymatically triggered 1,2-dioxetane chemiluminescence (6,7) for the detection, The principle of this method is illustrated in Fig. 1. The target DNA is immobilized onto a membrane and labeled with the enzyme alkaline phosphatase via an affinity system, such as the biotin/(strept)avidin or the digoxigenin system. Upon adding the dioxetane substrate to the membrane, the phosphatase will deprotect 1,2-dioxetane molecules by cleaving off the phosphate group, thereby initiating the chemiluminescent reaction that can then be detected as visible light. The main features of this detection system are speed, with detection times between l–30 min, sensitivity, between lo–100 attomoles of target DNA, and compatibility, since the method requires only equipment available in any molecular biology laboratory and allows the generation of the same data format (e.g., X-ray film hardcopy) as with radioactive detection. Since its first description in 1989 (3) several modifications and improvements have been reported for chemiluminescent detected DNA sequencing (7–11).
Fig. 1.

Schematic representation of chemiluminescent detection of membrane immobilized DNA.


Chemiluminescent Detection System Affinity System Plastic Wrap Radioactive Detection Enzyme Alkaline Phosphatase 
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  1. 1.
    Smrth, 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 inautomated DNA sequence analysis. Nature 321, 674–679.CrossRefGoogle Scholar
  2. 2.
    Beck, S (1987) Calorimetric detected DNA sequencing. Anal. Biochem 164, 514–520.PubMedCrossRefGoogle Scholar
  3. 3.
    Beck, S, O’Keeffe, T, Coull, J. M., and Kdster, H (1989) Chemiluminescent detectron of DNA: applications for DNA sequencing and hybridtzation Nucl. Acids Res. 17, 5115–5123PubMedCrossRefGoogle Scholar
  4. 4.
    Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chainterminating inhibitors. Proc Natl. Acad. Sci USA 74, 5463–5467PubMedCrossRefGoogle Scholar
  5. 5.
    Bankrer, A. T., Weston, K.M., and Barrell, B. G. (1987) Random cloning andsequencing. Meth Enzymol. 155, 5–93.Google Scholar
  6. 6.
    Schaap, A. P., Sandison, M.D., and Handley, R S. (1987) Chemical and enzymatte triggering of 1,Zdioxetanes alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane Tet Lett 28, 1159–1162CrossRefGoogle Scholar
  7. 7.
    Beck, S. and Koster, H. (1990) Applications of dioxetane chemiluminescent probes to molecular biology Anal Chem. 62, 2258–2270PubMedCrossRefGoogle Scholar
  8. 8.
    Trzard, R., Cate, R. L, Ramachandran, K L., Wysk, M., Voyta, J C, Murphy, 0.J., and Bronstein, I. (1990) Imagmg of DNA sequences with chemiluminescence. Proc Natl. Acad. Sci. USA 87, 4514–4518CrossRefGoogle Scholar
  9. 9.
    Chee, M. (1991) Enzymatic multiplex DNA sequencmg. Nucl Acids Res. 19, 3301–3305PubMedCrossRefGoogle Scholar
  10. 10.
    Creasey, A, D’Angro, L., Dunne, T S., Krssmger, C., O’Keeffe, T., Perry-O’Keefe, H., Moran, L. S, Roskey, M., Schildkraut, I., Sears, L. E., and Slatko, B. (1991) Apphcatton of a novel chemilummescence-based DNA method to single-vector and multiplex DNA sequencing Blotechniques 11, 102–109Google Scholar
  11. 11.
    Martin, C., Bresmck, L, Juo, R-R., Voyta, J C., and Bronstein, I. (1991) Improved chemilummescent DNA sequencing. Biotechmques 11, 110–113Google Scholar
  12. 12.
    Beck, S. (1993) Non-radioactrve detection of DNA using dioxetane chemrlummescence. Recombinant DNA, Part G, in Methods in Enzymology (Wu, R., ed ), Academic Press, Orlando, FL, vol 216, 143–153.Google Scholar

Copyright information

© Humana Press Inc. Totowa, New Jersey 1993

Authors and Affiliations

  • Stephan Beck
    • 1
  1. 1.Imperial Cancer Research FundLondonEngland

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