PCR Annealing Temperature Optimization

A polymerase chain reaction (PCR) uses a thermocycler to replicate target DNA templates and typically consists of three main stages including denaturing, annealing and extension. The annealing step is particularly important as this is the phase where primers bind to their complementary sequence on each single stranded (ss) DNA. These primer sets bracket a specific region of the DNA to be copied, known as the amplicon, to enable targeted extension by Taq polymerase in the following step. Though the annealing step is almost instantaneous, usually between 30 seconds to 1 minute, the PCR machine must drop to the appropriate annealing temperature (Ta) to enable effective primer-template binding. For this reason, optimizing the annealing temperature of the reaction is particularly important to allow for high primer-template fidelity and primer extension.

Tools:

Temperature of Annealing Calculator

The annealing temperature chosen for a given PCR relies directly on length and composition of the primers used. In general, an annealing temperature is roughly 3 - 5 °C below the melting temperature (Tm) of the primers is appropriate. Though an optimal annealing temperature should also take into consideration the melting temperature of the product. Rychlik et al. have previously defined an equation for determining an optimal annealing temperature:

Ta = (0.3 x (Tm of primer-template pair)) + (0.7 x (Tm of PCR product)) - 14.9

So first, optimization of annealing temperature must begin by determining the melting temperature values of the primer-template pair. A few common equations found throughout the literature are as follows:

For primers < 14 bp:   Tm = 4(G+C) + 2(A+T)

For primers > 13 bp:   Tm = 64.9 + (41 x (G+C-16.4) / (A+T+G+C))

Accounting for mismatch:   Tm = 81.5 + (0.41 x (%GC)) - (675 / (A+T+G+C)) - %mismatch

More complex formulas can also be used for primers of different length and variable nucleotide content (Sambrook et al. 1989; Sharrocks et al. 1994), though all calculated melting temperatures should simply be regarded as approximations since this parameter is variably affected by Mg2+ concentration, salt concentration, and even the primer and template concentrations in the reaction. Generally, optimizing the annealing temperature may take trial and error. It may be necessary to set the annealing temperature 5 °C below the Tm of the primer-template set, and perform multiple separate PCRs while increasing the annealing temperature in 1 - 2°C increments each time. Alternatively, the most common way of determining the melting temperature of the PCR product is by subjecting the product to a temperature gradient in the presence of intercalating dye that only emits light when bound to dsDNA. From here, a melting curve can be constructed to provide precise knowledge of the product's melting temperature.

Products

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References

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Optimization of the annealing temperature for DNA amplification in vitro.
Molecular cloning: a laboratory manual.
The Transcription Factors Elk-1 and Serum Response Factor Interact by Direct Protein-Protein Contacts Mediated by a Short Region of Elk-1
Optimization and troubleshooting in PCR