Design of oligonucleotide primers

Background and advice for designing primers, taken from the same book as the previous post.

  • Specificity is crucial; the longer, the higher specificity.
  • Probability: K = [g/2]^G+C x [(1-g)/2]^A+T — K is expected frequency, g is relative G+C content, GCAT number of each nucleotide.
  • 15 nucleotides would be unique within 3×10⁹ genome, but due to bias in codon usage, repetitive DNA sequences and gene families, they are not.
  • Good idea to scan DNA databases to see if only the wanted sequence is returned.

Selecting primers

  • Analysis: free of homopolymeric tracts, no secundary structures, not self-complimentary, no significant homology with other sequences.
  • List possible forward/reverse primers with calculating the melting point.
  • Selection of well-matched primers: similar in G+C content, but no more than 3 consecutive nucleotides should be complementary.
  • Refine length and placement of oligonucleotides: 3′-terminal nucleotide is G or C.

Primer design properties

Base composition

  • G+C between 40-60%.
  • Even distributions of all four bases along the length of the primer.


  • Complementary region should be 18-25 nucleotides.
  • Primer pair should not differ by >3bp.

Repeated and self complementary sequences

  • Inverted or self complementary should not be >3bp in length.
  • Tend to form hairpins and obstruct the annealing.

Complementary between primer pair

  • 3′ terminal should not bind to any site of the other one.
  • Hybrids formation will compete with the expected amplification.

Melting temperature

  • Temperature of primers (in pair) should not differ by >5°C.
  • Temperature of amplified product should not differ >10°C.
  • Ensures that denaturation occurs in each cycle.

3′ termini

  • Crucially should end with G or C.
  • However, GC or CG are not recommended since it increases the probability of forming hairpins.

Adding sequences to 5′ termini

  • Restriction sites, bacteriophage promoters, etc, commonly added to 5′ end.
  • Does not affect annealing.

Placement of priming sites

  • Maybe constrained by the location of mutations, restriction sites ,coding sequences, microsatellites, cis-acting elements.
  • When for cDNA best to use sequences that bind to different exons, so that contaminating genomic DNA is easily distinguished.

Primers for degenerate PCR

  • If short sequence aminoacid only, a pool of degenerate oligonucleotides containing all possible coding combinations can be used to amplify.