Direct DNA Sequencing of Polymerase Chain Reaction Products Using Magnetic Beads

  • Joakim Lundeberg
  • Bertil Pettersson
  • Mathias Uhlén
Part of the Springer Protocols Handbooks book series (SPH)


The use of magnetic particles in many fields of biochemistry, molecular biology, and medicine has been well documented and several magnetic particles are now available for diagnostic and cell separation purposes. The solid-phase approaches has improved robustness by the increased reproducibility with accompanying higher yields. Furthermore, automation has been facilitated, as reaction buffers and additional reagents can be rapidly changed without centrifugation or precipitation steps. The introduction of magnetic particles into DNA sequencing technology has proven to be an attractive alternative compared with traditional means of performing DNA sequencing.


Polymerase Chain Reaction Template Paramagnetic Bead Annealing Buffer Dilute Stock Solution Free Biotin 
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.


  1. 1.
    Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74(12), 5463–5467.PubMedCrossRefGoogle Scholar
  2. 2.
    Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233, 1076–1078.PubMedCrossRefGoogle Scholar
  3. 3.
    Hultman, T., Ståhl, S., Hornes, E., and Uhlén, M. (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17, 4937–4946.PubMedCrossRefGoogle Scholar
  4. 4.
    Hultman, T., Bergh, S., Moks, T., and Uhlen, M. (1991) Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. BioTechniques 10(1), 84–93.PubMedGoogle Scholar
  5. 5.
    Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of PCR-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440.PubMedCrossRefGoogle Scholar
  6. 6.
    Leren, T. P., Rødningen, O. K., Røsby, O., Solberg, K., and Berg, K. (1993) Screening for point mutations by semi-automated DNA sequencing using Sequenase and magnetic beads. BioTechniques 14(4), 618–623.PubMedGoogle Scholar
  7. 7.
    Khurshid, F. and Beck, S. (1993) Error analysis in manual and automated DNA sequencing. Anal. Biochem. 208, 138–143.PubMedCrossRefGoogle Scholar
  8. 8.
    Carothers, A. M., Urlaub, G., Mucha, J., Grunberger, D., and Chasin, L. A. (1989) Point mutation analysis in mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method. BioTechniques 7, 494–499.PubMedGoogle Scholar
  9. 9.
    Hedrum, A., Ponten, F., Ren, Z., Lundeberg, J., Pontén, J., and Uhlén, M. (1994) Sequence-based analysis of the human p53 gene based on microdissection of tumor biopsy samples. Biotechniques 17, 1–9.Google Scholar
  10. 10.
    Wahlberg, J., Albert, J., Lundeberg, J., Cox, S., Wahren, B., and Uhlen, M. (1992) Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT) treated patients. FASEB J. 6, 2843–2847.PubMedGoogle Scholar
  11. 11.
    Leitner, T., Halapi, E., Scarletti, G., Rossi, P., Albert, J., Fenyö, E.-M., and Uhlén, M. (1993) Analysis of heterogeneous viral populations by direct DNA sequencing. BioTechniques 15(1), 120–127.PubMedGoogle Scholar
  12. 12.
    Larder, B. A., Kohli, A., Kellam, P., Kemp, S. D., Kronick, M., and Henfrey, R. D. (1993) Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing. Nature 365, 671–673.PubMedCrossRefGoogle Scholar
  13. 13.
    Holmberg, A., Fry, G., and Uhlén, M. (1993) Automated DNA sequencing and analysis techniques, Venter, C, ed., Academic, London, pp. 139–145.Google Scholar
  14. 14.
    Wahlberg, J., Albert, J., Lundeberg, J., Fenyö, E.-M., and Uhlén, M. (1991) Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 2 variants by direct solid phase DNA sequencing. AIDS Res. Hum. Retrov. 7(12), 983–990.CrossRefGoogle Scholar
  15. 15.
    Wahlberg, J., Lundeberg, J., Hultman, T., and Uhlén, M. (1990) General colorimetric method for DNA diagnostics allowing direct solid phase genomic sequencing of the positive samples. Proc. Natl. Acad. Sci. USA 87, 6569–6573.PubMedCrossRefGoogle Scholar
  16. 16.
    Pettersson, B., Johansson, K.-E., and Uhlén, M. (1994) Sequence analysis of 16S rRNA from Mycoplasmas by direct solid-phase DNA sequencing. Appl. Env. Microbiol. 60(7), 2456–2461.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Joakim Lundeberg
    • 1
  • Bertil Pettersson
    • 1
  • Mathias Uhlén
    • 1
  1. 1.Department of Biochemistry, Kungliga Tekniska HogskolanRoyal Institute of TechnologyStockholmSweden

Personalised recommendations