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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Annealing Temperature (Ta)

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.
  • 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,
    1. Tm(primer) = Melting temperature of the primers
    2. 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 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.