Advertisement

Cycle Sequencing

  • Robert W. Blakesley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 167)

Abstract

Cycle sequencing is a variant of standard dideoxy, chain terminator DNA sequencing (1, 2, 3). The protocol has gained popularity owing to at least three features: simple execution, robust performance, and signal amplification. The main characteristic that defines the cycle sequencing protocol is that the sequencing reactions are incubated in a thermal cycler, with programs similar to those used in polymerase chain reaction (PCR). This method assures efficient, reproducible utilization of even difficult templates by repeated thermal denaturation of the DNA template during the sequencing reactions. In fact, through heat cycling the same template molecules are used repeatedly, resulting in accumulation of sufficient sequencing signal even when a very limited amount of template is used, for example, when sequencing DNA from single bacterial colonies or phage plaques (4,5). It should be made clear, however, that no new template molecules are created as they are in PCR; cycle sequencing products accumulate linearly, not geometrically, in this single-primer DNA synthesis reaction. Cycle sequencing can be performed using a variety of labeling and detection techniques. The following protocol utilizes radioactive end-labeled primer and X-ray film.

Keywords

Cycle Sequencing Template Molecule Cycle Sequencing Reaction Sequencing Buffer Generate Polymerase Chain Reaction Product 
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.

References

  1. 1.
    Murray, V. (1989) Improved double-stranded DNA sequencing using linear polymerase chain reaction. Nucleic Acids Res. 17, 8889.PubMedCrossRefGoogle Scholar
  2. 2.
    Craxton, M. (1991) Linear amplification sequencing, a powerful method for sequencing DNA. Methods Comp. Methods Enzymol. 3, 20–26.CrossRefGoogle Scholar
  3. 3.
    Blakesley. R. W. (1991) Cycle sequencing, in Methods in Molecular Biology, vol. 23: DNA Sequencing Protocols (Griffin, H.G. and Griffin, A. M., eds.), Humana Press, Totowa,NJ, pp. 209–217.Google Scholar
  4. 4.
    Krishnan, B. R., Blakesley, R. W., and Berg, D. E. (1991) Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res. 19, 1153.PubMedCrossRefGoogle Scholar
  5. 5.
    Young, A. and Blakesley, R. (1991) Sequencing plasmids from single colonies with the dsDNA Cycle Sequencing System. Focus 13, 137.Google Scholar
  6. 6.
    Pless, R. C. (1992) Cycle sequencing of DNA using 5′-33P-labeledprimers. Focus 14, 102–103.Google Scholar
  7. 7.
    Fan, J., Ranu, R. S., Smith, C., Ruan, C., and Fuller, C. W. (1996) DNA sequencing with [α α-33P]-labeled ddNTP terminators: A new approach to DNA sequencing with Thermo SequenaseTM DNA polymerase. BioTechniques 21, 1132–1137.PubMedGoogle Scholar
  8. 8.
    Tabor, S. and Richardson, C. C. (1995) A single residue in DNA polymerases of the Escherishia coli DNA polymerase I family is critical for distinguishing between deoxy-and dideoxynucleotides. Proc. Natl. Acad. Sci. USA 92, 6339–6343.PubMedCrossRefGoogle Scholar
  9. 9.
    Vander Horn, P. B., Davis, M. C., Cunniff, J. J., Ruan, C., McArdle, B. F., Samols, S. B., et al. (1997) Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing. BioTechniques 22, 758–762.PubMedGoogle Scholar
  10. 10.
    Meis, R. (1997) Increased sensitivity in DNA sequencing with the new SequiTherm EXCELTM II DNA polymerase. Epicentre Forum 4(2), 5.Google Scholar
  11. 11.
    Tabor, S. and Richardson, C. C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767–4771.PubMedCrossRefGoogle Scholar
  12. 12.
    Bankier, A. T. (1991) Electrophoresis of sequence reaction samples, in Methods in Molecular Biology, vol. 23: DNA Sequencing Protocols (Griffin, H. G. and Griffin, A. M., eds.), Humana Press, Totowa, NJ, pp. 121–129.Google Scholar
  13. 13.
    Innis, M. and Gelfand, D. (1990) Optimization of PCRs, in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic Press, San Diego, CA, pp. 3–12.Google Scholar
  14. 14.
    Khan, A. S., Wilcox, A. S., Hopkins, J. A., and Sikela, J. M. (1991) Efficient double stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers. Nucleic Acids Res. 19, 1715.PubMedCrossRefGoogle Scholar
  15. 15.
    Crouse, J. and Amorese, D. (1987) Ethanol precipitation: Ammonium acetate as an alternative to sodium acetate. Focus 9(2), 3–5.Google Scholar
  16. 16.
    Craxton, M. (1993) Cosmid sequencing, in Methods in Molecular Biology, vol. 23: DNA Sequencing Protocols (Griffin, H. G. and Griffin, A. M., eds.), Humana Press, Totowa, NJ, pp. 149–167.CrossRefGoogle Scholar
  17. 17.
    Krishnan, B. R., Kersulyte, D., Brikun, I., Berg, C. M., and Berg, D. E. (1991) Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of γ phage clones. Nucleic Acids Res. 19, 6177–6182.PubMedCrossRefGoogle Scholar
  18. 18.
    Adams, S. M. and Blakesley, R. W. (1992) Sequencing a PCR-ampli-fied DNA with the dsDNA Cycle Sequencing System. Focus 14, 31.Google Scholar
  19. 19.
    Gyllensten, U. B., Allen, M., and Josefsson, A. (1992) Sequencing of in vitro amplified DNA, in The PCR Technique: DNA Sequencing (Ellingboe, J., ed.), Eaton, Natick, MA, pp. 1–15.Google Scholar
  20. 20.
    Phear, G. A. and Harwood, J. (1994) Direct sequencing of PCR products, in Methods in Molecular Biology, vol. 31: Protocols for Gene Analysis (Harwood, A. J., ed.), Humana Press, Totowa, NJ, pp. 247–256.CrossRefGoogle Scholar
  21. 21.
    Brow, M. A. D. (1990) Sequencing with Taq DNA polymerase, in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic Press, San Diego, CA, pp. 189–196.Google Scholar
  22. 22.
    Paithankar, K. R. and Prasad, K. S. N. (1991) Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res. 19, 1346.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Robert W. Blakesley
    • 1
  1. 1.Molecular Biology Research & DevelopmentLife TechnologiesRockville

Personalised recommendations