Design recommendations

A good design of your oligonucleotides is of utmost importance, not just to a successful outcome of your assay but also to obtain high yields from the oligonucleotide manufacturer. The yield obtained from synthesis is to a certain extent sequence dependent. The four nucleotides dA, dG, dC and dT have different properties in terms of their ability to couple, detritylate and to sustain chemical exposure. How these nucleotides are combined in a sequence will therefore affect the synthesis yield.

Listed below are a few simple design guidelines to be considered.

General recommedations:

  • Keep the dG-dC content within 40-65% range if possible.
  • Please avoid long runs of a single base. Especially dAs and dGs.
  • If possible, start out by designing your probes, and then consider where to locate your primers.

Primer design recommendations:

  • The melting temperature of the primers should be 52-58°C. Primers with melting temperature above 65°C have a tendency for secondary annealing.
  • The presence of dG or dC bases within the last five bases from the 3'-end of the primers (GC clamp) helps to promote specific binding at the 3'-end, due to the stronger bonding of dG and dC bases. However, more than three dGs or dCs should be avoided in the last five bases at the 3'-end.
  • Avoid dG at the 3'-end.

Probe design recommendations:

  • The melting temperature of the probe should be 8-12°C higher than the melting temperature of the primers used for amplification.
  • Avoid dG at the 5'-end. dG acts as a weak quencher and should be separated from the dye with at least four other nucleotides.
  • Try to avoid multiples of identical nucleotides. This is especially important for dG, where four or more dGs in a row definitely should be avoided.
  • Choose the strand that gives the probes more dCs than dGs.
  • Should the probe be longer than 25 nucleotides, use an internal quencher positioned 7-14 nucleotides apart from the reporter dye.