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Secugen - Technical Solutions - Double Sequence

Sequence Appearance.

  • Double peaks are observed. Multiple peaks with the same or different height overlapping one another.
  • Raw data has adequate signal intensity, indicating that the DNA sequencing reaction yielded product, so the double peaks are not due to weak signal and/or background noise.


Primer Dimer


Primer Dimer

Clone Contamination

Two Primers

Clone Contamination
Two Primers

Possibles Causes.

  • PCR products were not purified (or the purification was not performed properly). In this case, residual PCR primers or primer dimers may participate in the sequencing reaction.
  • PCR template may be heterozygous due to indels present in a diploid (or polyploid) organism.
  • Clone contamination. In this case, the beginning of the sequence is often clean and becomes double as the sequence progresses into the two inserts.
  • Two primers may have been mistakenly added to the sequencing reaction.
  • The PCR reaction yields more than a product, and both are sequenced.
  • There may be another region in the molecule with enough homology to the primer.


  • If clone contamination is expected, please plate again the bacteria onto selection media, and select clones again.
  • Where PCR template heterozygosity is suspected, examination of the ‘dirty’ sequence may yield some indication of the area of heterozygosity. Newly designed PCR primers that yield shorter products may allow you to discern where the troublesome area is. Alternatively, you may decide to redesign the sequencing primer close to the area where the problem first arises hoping to find a primer that will sequence one of the product species. If the PCR product is large, subcloning the template into shorter pieces may also provide a strategy for discerning the true sequence of the area of interest.
  • Run the PCR reaction in an agarose gel in order to check if more than one amplicon is present.
  • If you suspect that two different primers have been added to the reaction, repeat the reaction. If the working solution for the sequencing primer may be contaminated then going back to your original uncontaminated stock solution to prepare a new working solution or resynthesize the primer.
  • Check your PCR purification protocol and the solutions used in the clean-up reaction.
  • Check the primer sequence against the template sequence to ensure that there is a single binding site. Where sequence is unknown, you may need to switch to a different sequencing primer to eliminate the problem.