4.9. Assay optimization

Each assay should be optimized experimentally since the various components can significantly affect the reaction dynamics (Caetano-Anolles, 1998). The criteria for optimization can be sensitivity, specificity or reproducibility, and for qPCR also reaction efficiency. Higher primer concentrations and lower annealing temperatures increase sensitivity, but reduce specificity. Optimising for reproducibility usually means identifying the highest annealing temperature, the lowest primer concentrations and the shortest incubation times that consistently generate the right product, without secondary products, at a consistent amplification cycle.