Primer design guide

Primers (also: oligos or oligonucleotides) are short sequences of nucleotides used for PCR-based DNA amplification. We use them for strain genotyping, molecular cloning, and sequencing. There are several characteristics we seek when designing primers:

  1. Software tool to use
    We usually design primers by loading a DNA sequence of interest into Snapgene and simply selecting a stretch of DNA with the computer mouse. Snapgene automatically computes the melting temperature of a selected DNA sequence and displays it near the top of the window.
  2. Melting temperature
    Primers should have a melting temperature between 50°C and 60°C. Most of the primers we use in the lab have a melting temperature of Tm = 56°C. Aim for the same Tm for your primers.
  3. Choose at least one C or G at the 3′ end
    Because DNA polymerase adds nucleotides to the 3′ end of the primer, it is common to choose C’s and G’s as the last nucleotide(s) of the primer because the C-G bond is stronger than the A-T bond, and may create a more stable end for the DNA polymerase to extend.
  4. GC content
    Primers should have a GC content of around 50% (roughly the same amount of GC and AT nucleotides). Deviations from this of up to 20% are usually tolerated.
  5. Length
    A primer should be between 15 bp and 25 bp long.
  6. Repeats
    A primer should not contain more than three consecutive nucleotides that are the same.
  7. Uniqueness
    A pair of primers should define a unique PCR product in the template DNA. Random DNA sequences are essentially unique but be careful when you are working with repetitive sequences.
  8. Secondary structure
    A primer should not have a strong secondary structure (example: ..GATATA..TATATC.. can form a DNA hairpin). They can be hard to catch by just looking at the sequence and you can check for their presence using online tools. (In practice, most of us do not really check for secondary structure, although we should.)
  9. Primer-dimer
    A primer-dimer is an undesired by-product of the PCR reaction which comes about when the two primers are partly complementary. To avoid it, you should not use pairs of primers that can bind to each other.

Author: Vojislav Gligorovski
December 2021