Molecular Biotechnology

, Volume 10, Issue 2, pp 183–185 | Cite as

Direct sequencing of long polymerase chain reaction fragments



Direct sequencing of polymerase chain reaction (PCR)-generated templates is a commonly used technique in molecular biology laboratories. We describe an improved method for direct sequencing of PCR fragments longer than 20 kb obtained with a commercial mixture ofTaq andPwo DNA polymerases. The sequencing protocol was optimized for an automated infrared DNA sequencer, consistently yielding long reads (500–600 bases).

Index Entries

Primer walking DNA purification automated DNA sequencing PCR 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  2. 2.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1997)Current Protocols in Molecular Biology. Wiley, New York.Google Scholar
  3. 3.
    Chen, J. D., and Morrison, D. A. (1987) Cloning ofStreptococcus pneumoniae DNA fragments inEscherichia coli requires vectors protected by strong transcriptional terminators.Gene 55, 179–187.PubMedCrossRefGoogle Scholar
  4. 4.
    Martin, B., Alloing, G., Boucraut, C., and Claverys, J. P. (1989) The difficulty of cloningStreptococcus pneumoniae mal andami loci inEscherichia coli: toxicity ofmalX andamiA gene products.Gene 80, 227–238.PubMedCrossRefGoogle Scholar
  5. 5.
    Benes, V., Kilger, C., Voss, H., Paabo, S., and Ansorge, W. (1997) Direct primer walking on P1 plasmid DNA.BioTechniques 23, 98–100.PubMedGoogle Scholar
  6. 6.
    Middendorf, L. R., Bruce, J. C., Bruce, R. C., Eckles, R. D., Grone, D. L., Roemer, S. C., Sloniker, J. D., Steffesen, D. L., Sutter, S. L., Brumbaugh, J. A., and Patonay, G. (1992) Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus.Electrophoresis 13, 487–494.PubMedCrossRefGoogle Scholar
  7. 7.
    Hynes, W. L., Ferretti, J. J., Gilmore, M. S., and Segarra, R. A. (1992) PCR amplification of streptococcal DNA using crude cell lysates.FEMS 94, 139–142.CrossRefGoogle Scholar
  8. 8.
    Joshi, A. K., Baichwal, V., and Ferro-Luzzi Ames, G. (1991) Rapid polymerase chain reaction amplification using intact bacterial cells.BioTechniques 10, 42–44.PubMedGoogle Scholar
  9. 9.
    Sarkar, G. and Sommer, S. S. (1990) Shedding light on PCR contamination.Nature 343, 27.PubMedCrossRefGoogle Scholar
  10. 10.
    Lindahl, T. (1993) Instability and decay of the primary structure of DNA.Nature 362, 709–715.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1998

Authors and Affiliations

  • Francesco Iannelli
    • 1
  • Laura Giunti
    • 1
  • Gianni Pozzi
    • 1
  1. 1.Sezione di Microbiologia, Dipartimento di Biologia MolecolareUniversità di SienaSienaItaly

Personalised recommendations