The gene of interest usually has to be amplified from genomic or vector DNA by PCR (polymerase chain reaction) before it can be cloned into an expression vector. The first step is the design of the necessary primers.

Important features are:

Primer sequence. Especially the 3'-end of the primer molecule is critical for the specificity and sensitivity of PCR. It is recommended not to have:

  • 3 of more G or C bases at this position. This may stabilize nonspecific annealing of the primer.
  • a 3' thymidine, since it is more prone to mispriming than the other nucleotides.

Primer pairs should be checked for complementarity at the 3'-end. This often leads to primer-dimer formation.

Bases at the 5'-end of the primer are less critical for primer annealing. Therefore, it is possible to add sequence elements, like restriction sites, to the 5'-end of the primer molecule.

Primer length. Usually a primer length of 18-30 bases is optimal for most PCR applications. Shorter primers could lead to amplification of nonspecific PCR products.

Melting temperature (Tm). The specificity of PCR depends strongly on the melting temperature (Tm) of the primers (the temperature at which half of the primer has annealed to the template). Usually good results are obtained when the Tm's for both primers are similar (within 2-4 °C) and above 60°C. The Tm for a primer can be estimated using the following formula:

Tm = 2°C * (A + T) + 4°C * (C + G)

GC content. The GC content of a primer should be between 40 and 60%.

Design of the 5'-end primer

The 5'-end primer overlaps with the 5'-end of the gene of interest and should contain the following elements:

  • Restriction site. The restriction site should be the same or provide the same sticky end to the first of the restriction enzymes in the multiple cloning site of the vector chosen to clone the gene of interest into. Alternatively, you could pick any restriction enzyme that gives a blunt end upon cleavage (see cloning). Often Nco ICCATGG) or Nde I (CATATG) are chosen because the ATG within these sites can be used directly to create the ATG start codon and/or the ATG codon for the N-terminal methionine residue (see Utilisation of the Nco I cloning site)
  • 5'-extension to the restriction site. Restriction enzymes cleave DNA much less efficient towards the end of a fragment. A 5' extension of the restriction site with 2-10 bases greatly increases the cleavage efficiency of most enzymes. Data on the effect of the extension length and sequence on the cleavage efficiencies of the most used restriction enzymes can be found in the reference appendix of the New England Biolabs catalogue.
  • Start codon. A start codon (usually ATG) should be included when the gene of interest is not expressed with an N-terminal tag or fusion partner or when an N-terminal methionine residue is present. It should be checked that the start codon and the gene of interest are in frame with an eventual N-terminal tag and/or fusion partner.
  • Overlap with the gene of interest. The overlap between the primer and the gene of interest should be long enough to give a Tm of 60°C or more (calculated as shown above).

Design of the 3'-end primer

The 3'-end primer overlaps with the DNA strand complementory to the 3'-end of the gene of interest and should contain the following elements:

  • Restriction site. The restriction site should be the same or provide the same sticky end to the second of the restriction enzymes in the multiple cloning site of the vector chosen to clone the gene of interest into. Alternatively, you could pick any restriction enzyme that gives a blunt end upon cleavage (see cloning)
  • 5'-extension to the restriction site. Restriction enzymes cleave DNA much less efficient towards the end of a fragment. A 5' extension of the restriction site with 2-10 bases greatly increases the cleavage efficiency of most enzymes. Data on the effect of the extension length and sequence on the cleavage efficiencies of the most used restriction enzymes can be found on pp. 210-211 of the 2000/01 catalog of New England Biolabs.
  • Stop codon(s). A stop codon (TAA is preferred because it is less prone to read-through than TAG and TGA) should be included when no C-terminal tag is used. To increase the efficiency of termination it is possible to use 2 or 3 stop codons in series.
  • Overlap with the stand complement to the 3'-end of the gene of interest. The overlap between the primer and the strand complement to the 3'-end of the gene of interest should be long enough to give a Tm of 60°C or more (calculated as shown above). It should be checked that the gene of interest is in frame with an eventual C-terminal tag.

Example: Primer design for the PCR amplification of the coaD gene