Traditional PCR cycles include denaturation, annealing and extension steps but in real-time PCR, annealing and extension are merged into one step resulting in two steps per cycle. This is because of short (typically 75-150 bp) amplicon length and the power of Taq polymerase used in real-time PCR. Long (>400 bp) amplicons or primers with annealing temperatures <60°C may still require a separate extension step (3-step protocol) for optimum polymerase performance.