DNA Sequencing Traces With Primer Dimers
Identification of Primer Dimer Problems in Traces
- The trace signal is mixed in the early regions (normally before base 400) yet the late regions are not mixed (Figure 1).
- The signal drops from very high early in the trace (usually in the first 100 bases) to much lower levels. Examination of the raw signal shows a high signal "block" (Figure 2).
- The total quality scores counts are generally low to moderate depending on the region affected.
Figure 1. Example of a primer dimer problem in the sequencing reaction. (A) Mixed signal Early region of the trace . (B) Readable sequence in the late regions of the trace.
Figure 2. A high peak signal "block".
Causes of Primer Dimers in Sequencing Reactions
- Contamination of the template, primer stock or other sequencing reagents with primer dimers.
- Too low an annealing temperature.
- Two primer binds site present.
- Sequencing TempliPhi templates. For reasons unknown TempliPhi templates are much more prone to primer dimer problems.
Solving Primer Dimer Problems
- Change all the sequencing reagents and water for a fresh batch. Also clean the pipettors as they can also be the cause of the primer dimer contamination.
- Raise the annealing temperature and/or use a shorter annealing time.
- Check the sequence of the vector to ensure that two primer binding sites are not present. Be especially careful of partial matches (8-9 base pair match can cause problems if at the 3' end of the primer).
For more information on detecting DNA sequencing trace problems please visit the QualTrace DNA sequencing analysis software page.
Return to the main DNA sequencing troubleshooting page.