Ta = 0.3 × Tm(primer) + 0.7 × Tm(product) − 14.9
The annealing temperature (Ta) is critical for PCR specificity and yield. Too high → no amplification; too low → non-specific products. The simplest estimate: Ta = Tm − 5°C. For short primers (<14 bp): Tm = 2(A+T) + 4(G+C) (Wallace rule). For longer primers: Tm = 64.9 + 41(nG+nC−16.4)/length. When primers have different Tm values, use the lower one minus 5°C, or optimize with gradient PCR. Touch-down PCR starts at high Ta and decreases each cycle to improve specificity. Modern polymerases with improved processivity may allow higher Ta. Always verify your Ta experimentally with positive and negative controls.