Standard Article

Direct Sequencing of PCR Products

  1. S Wilton

Published Online: 24 OCT 2002

DOI: 10.1038/npg.els.0003769

eLS

eLS

How to Cite

Wilton, S. 2002. Direct Sequencing of PCR Products. eLS.

Author Information

  1. Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australia

Publication History

  1. Published Online: 24 OCT 2002

Abstract

Direct sequencing of PCR products allows immediate and detailed analysis of those particular fragments without the extra steps associated with cloning. Although cloning of PCR products is now routine, there are still extra steps (which may take several days) in obtaining the recombinant sequencing templates. In addition to this extra effort, it is then necessary to sequence at least four independent recombinants to ensure that the average sequence has been obtained, so as to detect differences in the cloned sequences reflecting errors introduced during the PCR. Prior to the introduction of the thermostable polymerases and cycle sequencing, double-stranded sequencing of either PCR products or plasmid DNA was less efficient and more difficult than sequencing single-stranded templates. Fluorescent sequencing chemistry and template preparation have advanced to such a state where it is now possible to generate hundreds of bases of sequence information from the same primers used in the original PCR. Further sequence information can be obtained using ‘internal’ primers.

Keywords:

  • DNA sequencing;
  • PCR products;
  • template purification;
  • chromatogram;
  • single tube reaction