Sanger DNA Sequencing: Primer Design

Proper primer design is one of the single most important factors in successful automated Sanger DNA Sequencing. Good sequencing results require high quality primers, just as much as high quality templates.

The following criteria are considered most critical in sequencing primer design:

  • Primer length should be in the range of 18 and 24 bases.
  • The primer should have a GC content of about 45-55%.
  • The primers should have a GC-lock (or GC "clamp") on the 3' end (i.e. the last 1 or 2 nucleotides should be a G or C residue).
  • The primer should have a melting temperature (Tm) greater than 50°C but less than 65°C.
  • The primer should not include homopolymeric runs of more than 4-5 nucleotides.
  • Avoid primers with secondary structures or the potential to self-hybridize.
  • Avoid designing primer upstream of homopolymeric or heteropolymeric regions (A, C, G or T repeats).
  • Check primer for specificity in annealing to template (= lack of secondary priming site).
  • Primer should be located at least 50-60 bases upstream of your sequence of interest.

Please note: No set of guidelines will always accurately predict the success of a primer. Some primers may fail for no apparent reason, and primers that appear to be poor candidates may work well for automated sequencing.

Below are links that offer free online primer design and analysis software:

Primer Design:

Primer Analysis: