Primer annealing temperature (Ta)—The primer melting temperature (Tm) estimates the DNA-DNA hybrid stability and is critical in determining the primer annealing temperature (Ta).
The Ta is necessary and critical for primer-template hybridization to occur. The annealing temperature (Ta) chosen for PCR relies on the length and composition of the primers. Generally, an annealing temperature should be approximately 10-150 C lower than the Tm.
- Too high Ta will produce insufficient primer-template hybridization, resulting in low PCR product yield. Too low Ta may lead to non-specific products caused by many base pair mismatches. Mismatch tolerance is found to have the most decisive influence on PCR specificity.
- Formula for calculating Ta: Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9 where,
- Tm(primer) = Melting temperature of the primers
- Tm(product) = Melting temperature of the product
- In general, it is routine to use an annealing temperature (Ta) of 10 to 15°C lower than the Tm.
Primer length - It is generally accepted that the optimal size of PCR primers is 18-22 base pairs (bp). This is long enough for adequate specificity and short enough for primers to bind efficiently to the template at the annealing temperature.
