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. 1990 Jun;87(12):4514–4518. doi: 10.1073/pnas.87.12.4514

Imaging of DNA sequences with chemiluminescence.

R Tizard 1, R L Cate 1, K L Ramachandran 1, M Wysk 1, J C Voyta 1, O J Murphy 1, I Bronstein 1
PMCID: PMC54146  PMID: 2191292

Abstract

We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert [Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995]. Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins [Church, G. M. & Kieffer-Higgins, S. (1988) Science 240, 185-188].

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ansorge W., Sproat B. S., Stegemann J., Schwager C. A non-radioactive automated method for DNA sequence determination. J Biochem Biophys Methods. 1986 Dec;13(6):315–323. doi: 10.1016/0165-022x(86)90038-2. [DOI] [PubMed] [Google Scholar]
  2. Ansorge W., Sproat B., Stegemann J., Schwager C., Zenke M. Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4593–4602. doi: 10.1093/nar/15.11.4593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beck S. Colorimetric-detected DNA sequencing. Anal Biochem. 1987 Aug 1;164(2):514–520. doi: 10.1016/0003-2697(87)90526-4. [DOI] [PubMed] [Google Scholar]
  4. Beck S., O'Keeffe T., Coull J. M., Köster H. Chemiluminescent detection of DNA: application for DNA sequencing and hybridization. Nucleic Acids Res. 1989 Jul 11;17(13):5115–5123. doi: 10.1093/nar/17.13.5115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bronstein I., Voyta J. C., Edwards B. A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Anal Biochem. 1989 Jul;180(1):95–98. doi: 10.1016/0003-2697(89)90093-6. [DOI] [PubMed] [Google Scholar]
  7. Brumbaugh J. A., Middendorf L. R., Grone D. L., Ruth J. L. Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5610–5614. doi: 10.1073/pnas.85.15.5610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Church G. M., Kieffer-Higgins S. Multiplex DNA sequencing. Science. 1988 Apr 8;240(4849):185–188. doi: 10.1126/science.3353714. [DOI] [PubMed] [Google Scholar]
  10. Edwards B., Sparks A., Voyta J. C., Bronstein I. Unusual luminescent properties of odd- and even-substituted naphthyl-derivatized dioxetanes. J Biolumin Chemilumin. 1990 Jan-Mar;5(1):1–4. doi: 10.1002/bio.1170050102. [DOI] [PubMed] [Google Scholar]
  11. Elder J. K., Green D. K., Southern E. M. Automatic reading of DNA sequencing gel autoradiographs using a large format digital scanner. Nucleic Acids Res. 1986 Jan 10;14(1):417–424. doi: 10.1093/nar/14.1.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gebeyehu G., Rao P. Y., SooChan P., Simms D. A., Klevan L. Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA. Nucleic Acids Res. 1987 Jun 11;15(11):4513–4534. doi: 10.1093/nar/15.11.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiraoka Y., Sedat J. W., Agard D. A. The use of a charge-coupled device for quantitative optical microscopy of biological structures. Science. 1987 Oct 2;238(4823):36–41. doi: 10.1126/science.3116667. [DOI] [PubMed] [Google Scholar]
  14. Jablonski E., Moomaw E. W., Tullis R. H., Ruth J. L. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986 Aug 11;14(15):6115–6128. doi: 10.1093/nar/14.15.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klotz I. M., Royer G. P., Sloniewsky A. R. Macromolecule--small molecule interactions. Strong binding and cooperativity in a model synthetic polymer. Biochemistry. 1969 Dec;8(12):4752–4756. doi: 10.1021/bi00840a015. [DOI] [PubMed] [Google Scholar]
  16. Klotz I. M., Sloniewsky A. R. Macromolecule-small molecule interactions: a synthetic polymer with greater affinity than serum albumin for small molecules. Biochem Biophys Res Commun. 1968 May 10;31(3):421–426. doi: 10.1016/0006-291x(68)90493-2. [DOI] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  19. Prober J. M., Trainor G. L., Dam R. J., Hobbs F. W., Robertson C. W., Zagursky R. J., Cocuzza A. J., Jensen M. A., Baumeister K. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 1987 Oct 16;238(4825):336–341. doi: 10.1126/science.2443975. [DOI] [PubMed] [Google Scholar]
  20. Rubin C. M., Schmid C. W. Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic Acids Res. 1980 Oct 24;8(20):4613–4619. doi: 10.1093/nar/8.20.4613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith L. M., Sanders J. Z., Kaiser R. J., Hughes P., Dodd C., Connell C. R., Heiner C., Kent S. B., Hood L. E. Fluorescence detection in automated DNA sequence analysis. Nature. 1986 Jun 12;321(6071):674–679. doi: 10.1038/321674a0. [DOI] [PubMed] [Google Scholar]
  23. Whitehead T. P., Kricka L. J., Carter T. J., Thorpe G. H. Analytical luminescence: its potential in the clinical laboratory. Clin Chem. 1979 Sep;25(9):1531–1546. [PubMed] [Google Scholar]
  24. Wick R. A. Photon counting imaging: applications in biomedical research. Biotechniques. 1989 Mar;7(3):262–269. [PubMed] [Google Scholar]

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