Primer annealing temperature (Ta) - The primer melting temperature (Tm) is the estimate of the DNA-DNA hybrid stability and critical in determining the primer annealing temperature (Ta).
The Ta is important and critical for the 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 possibly lead to non-specific products, caused by a high number of base pair mismatches. Mismatch tolerance is found to have the strongest 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
Primer length - It is generally accepted that the optimal length of PCR primers is 18-22 base pairs (bp). This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature
- In general, it is routine to use an annealing temperature (Ta) of 10 to 15°C lower than the Tm.