Direct Sequencing of PCR Products


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

Figure 1.

Direct sequencing chromatogram of a PCR product, 235 bp long. The abrupt drop‐off in signal corresponds to the end of the PCR product.

Figure 2.

Summary of the QIAquick clean‐up method.



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Wilton, S(Oct 2002) Direct Sequencing of PCR Products. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0003769]