Why does amplification efficiency decrease or plateau in the late cycles?
Three limitations may contribute to the decrease in amplification efficiency: • Plateau occurs in PCR because of the progressive reduction in the efficiency of the primer-template complex formation due to product reannealing. The primers in PCR are always present at high excess and the kinetics of reannealing are determined by the molecule in the greatest concentration. Typically, total initial PCR primer concentrations are 2×10-6 M. However, as the reaction proceeds, the DNA target concentration increases and the primer concentration decreases, albeit only slightly, to 1.4×10-7 M to 2×10-6 M, and it takes a longer time for the primer-template complex to form. If the product strands have a chance to reanneal to themselves, forming a very stable complex, the primers will not have a binding site for extension and doubling may not occur. This snap-back of product strands reannealing to themselves can occur in a few seconds (or less) at high DNA concentrations. • Primer concentrationdeplet
