Dideoxy DNA Sequencing with Chemiluminescent Detection

Chris S. Martin1

1 Tropix, Inc., Bedford, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.4B
DOI:  10.1002/0471142727.mb0704bs47
Online Posting Date:  May, 2001
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Abstract

Standard dideoxy DNA sequencing can be performed easily and efficiently with nonisotopic, chemiluminescent detection by utilizing primers labeled with biotin in the sequencing reactions. As described in this unit, reaction products are separated by denaturing gel electrophoresis, transferred to a nylon membrane, and detected by first binding a streptavidin‐alkaline phosphatase conjugate, then incubating with a chemiluminescent 1,2‐dioxetane substrate. The emitted light signal is imaged on standard X‐ray film, producing high‐resolution DNA sequencing ladders. Indirect alkaline phosphatase‐labeling of biotinylated DNA with free streptavidin and biotinylated alkaline phosphatase is also detailed, Finally, the detection of sequencing reactions labeled with other haptens using specific antibody‐alkaline phosphatase conjugates is described.

     
 
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Table of Contents

  • Basic Protocol 1: DNA Sequencing Using Biotinylated Primers with Chemiluminescent Detection
  • Alternate Protocol 1: Two‐Step (Indirect) Detection Using Streptavidin and Biotinylated Alkaline Phosphatase
  • Alternate Protocol 2: Sequencing with Hapten‐Labeled Primers and Detection with Antibody‐Alkaline Phosphatase Conjugates
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Sequencing Using Biotinylated Primers with Chemiluminescent Detection

  Materials
  • 1 µg single‐stranded DNA (∼0.5 pmole) or 1 to 3 µg denatured double‐stranded DNA template
  • Biotinylated DNA sequencing primer
  • Bst reaction buffer: 100 mM Tris˙Cl, pH 8.5 ( appendix 22)/100 mM MgCl 2
  • recipePrimer termination mixes (A, C, G, T; see recipe)
  • 1 U/µl Bst polymerase (Bio‐Rad)
  • 20× chase solution: 10 mM each of dATP, dCTP, dGTP, dTTP in H 2O
  • recipeStop solution (see recipe)
  • 0.2‐ to 0.4‐mm‐thick sequencing gel containing 8 M urea (unit 7.6)
  • TBE buffer ( appendix 22)
  • recipeBlocking buffer I (see recipe)
  • recipeConjugate solution (see recipe)
  • recipeWash buffer I (see recipe)
  • recipeAssay buffer (see recipe)
  • recipeDioxetane detection solution (see recipe)
  • 65°, 70°, and 80°C water baths or heating blocks or thermal cycler
  • Filter paper (Whatman 3MM or equivalent)
  • Nylon membrane (Table 7.4.1)
  • UV light source: UV cross‐linking apparatus, UV transilluminator, or hand‐held UV lamp
  • Large heat‐sealable bags (∼40 × 53–cm; Table 7.4.1)
  • X‐ray film (Kodak XAR‐5 or equivalent)
  • Additional materials for DNA sequencing gel electrophoresis (unit 7.6) and exposure and processing of X‐ray films ( appendix 3A)

Alternate Protocol 1: Two‐Step (Indirect) Detection Using Streptavidin and Biotinylated Alkaline Phosphatase

  • recipeTwo‐step blocking solution (see recipe)
  • recipeStreptavidin solution (see recipe)
  • recipeTwo‐step wash solution I (see recipe)
  • recipeBiotinylated alkaline phosphatase solution (see recipe)
  • recipeTwo‐step wash solution II (see recipe)

Alternate Protocol 2: Sequencing with Hapten‐Labeled Primers and Detection with Antibody‐Alkaline Phosphatase Conjugates

  • 5′‐hapten‐end‐labeled primer
  • recipeBlocking buffer II (see recipe)
  • recipeWash buffer II (see recipe)
  • recipeAntibody‐conjugate solution (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Beck, S., O'Keeffe, T., Coull, J.M., and Koster, H. 1989. Chemiluminescent detection of DNA: Application for DNA sequencing and hybridization. Nucl. Acids Res. 17:5115‐5123.
   Bronstein, I., Juo, R.‐R., Voyta, J.C., and Edwards, B. 1991. Novel chemiluminescent adamantyl 1,2‐dioxetane enzyme substrates. In Bioluminescence and Chemiluminescence: Current Status, (P. Stanley and L.J. Kricka, eds.) pp. 73‐82. John Wiley & Sons, Chichester.
   Church, G.M. and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991‐1995.
   Church, G.M. and Kieffer‐Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185‐188.
   Creasey, A., D'Angio Jr., L., Dunne, T.S., Kissinger, C., O'Keeffe, T., Perry‐O'Keeffe, H., Moran, L.S., Roskey, M., Schildkraut, I., Sears, L.E., and Slatko, B. 1991. Application of a novel chemiluminescence‐based DNA detection method to single‐vector and multiplex DNA sequencing. BioTechniques 11:102‐109.
   Höltke, H.J., Sanger, G., Kessler, C., and Schmitz, G. 1992. Sensitive chemiluminescent detection of digoxigenin‐labeled nucleic acids: A fast simple protocol and its applications. BioTechniques 12:104‐113.
   Karger, A.E., Weiss, R., and Gesteland, R.F. 1992. Electronic chemiluminescence imaging of DNA sequencing blots using a charge‐coupled device camera. Nucl. Acids Res. 20 (24):6657‐6665.
   Karger, A.E., Weiss, R., and Gesteland, R.F. 1993. A line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots. Anal. Chem. 65:1785‐1793.
  Kricka, L.J. (ed.) 1992. Nonisotopic DNA Probe Techniques. Academic Press, San Diego.
   Lakey, N.D., Ghazizadeh, H., Jaehn, L., Richterich, P., Robinson, K., and Church, G. 1993. Automated multiplex sequencing of E. coli genomic DNA. Genome Science and Technology I, p. 41.
   Martin, C., Bresnick, L., Juo, R.‐R., Voyta, J.C., and Bronstein, I. 1991. Improved chemiluminescent DNA sequencing. BioTechniques 11:102‐109.
   Mathews, J.A., Batki, A., Hynds, C., and Kricka, L.J. 1985. Enhanced chemiluminescent method for the detection of DNA dot‐hybridisation assays. Anal. Biochem. 151:205‐209.
   Olesen, C.E.M., Martin, C.S., and Bronstein, I. 1993. Chemiluminescent DNA sequencing with multiplex labeling. BioTechniques 15:480‐485.
   Pohl, F.M. and Beck, S. 1987. Direct transfer electrophoresis used for DNA sequencing. Methods Enzymol. 155:250‐259.
   Pollard‐Knight, D., Read, C.A., Downes, M.J., Howard, L.A., Leadbetter, L.A., Pheby, S.A., McNaughton, E., Syms, A., and Brady, M.A.W. 1990. Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase. Anal. Biochem. 185:84‐89.
   Prober, J.M., Trainor, G.L., Dam, R.J., Hobbs, F.W., Robertson, C.W., Zagursky, R.J., Cocuzza, A.J., Jensen, M.A., and Baumeister, K. 1987. A system for rapid DNA sequencing with fluorescent chain‐terminating dideoxynucleotides. Science 238:336‐341.
   Richterich, P. and Church, G.M. 1993. DNA sequencing with direct transfer electrophoresis and nonradioactive detection. Methods Enzymol. 218:187‐223.
   Richterich, P., Heller, C., Wurst, H., and Pohl, F.M. 1989. DNA sequencing with direct blotting electrophoresis and colorimetric detection. BioTechniques 5:52‐59.
   Tizard, R., Cate, R.L., Ramachandran, K.L., Wysk, M., Voyta, J.C., Murphy, O.J., and Bronstein, I. 1990. Imaging of DNA sequences with chemiluminescence. Proc. Natl. Acad. Sci. U.S.A. 87:4514‐4518.
Key References
   Creasey et al., 1991. See above.
  Describes the two‐step (indirect) detection protocol.
   Martin et al., 1991. See above.
  Describes the basic chemiluminescent detection protocol.
   Olesen et al., 1993. See above.
  Describes the basic chemiluminescent detection protocol, as well as the hapten‐labeled primer and antibody‐conjugate detection methods.
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