Resources | Primers | Primer Design

The type of primer that can be used for automated DNA sequencing is very dependent on the chemistry used. Since we use cycle sequencing for all our reactions, the primers must be designed with this in mind. Following a few basic rules will ensure that your primers have the correct properties for cycle sequencing:

  1. Always design your primers using known/good quality sequence
  2. Aim for a Tm of about 60 degrees Celsius [Tm=(sum A+T) x 2 + (sum G+C) x 4]
  3. Try to have about 50-55% GC content
  4. Have at least one G or C at the 3' end of the primer (but not GGG, etc.)
  5. Avoid homopolymeric regions (e.g. CCCCC)
  6. Avoid repeats of sequences (e.g. GATCGATC)
  7. Avoid any self-complementary regions (e.g. GATCNNNGATC)

The best-practices outlined above will certainly be familiar to all those who have routinely designed PCR primers. Cycle sequencing is similar to PCR (except only ONE primer is used) and so often primers that work for PCR will work for cycle sequencing.



  • PCR primers with excessively high Tms (over 65 Deg. C) may generate high backgrounds, when used for cycle sequencing.
  • Primers with low Tms (below 55 Deg. C) often give weak sequence.
    • This can also be seen with certain "standard" sequencing primers such as T3 and SP6.
      • For this reason these primers have had to be altered for automated sequencing.

Customers may wish to make use of various primer design software and services or may wish us to assist them in design of primers. Please note that we do charge for designing sequencing primers (please enquire). Whilst we do not recommend any particular design software, a basic free online design service that produces good output is available from Chang Bioscience.

Do you need any help? Please get in touch and we’ll be happy to lend a hand.