PCR has historically been performed as an endpoint assay, where
DNA is amplified then analyzed by gel electrophoresis. However,
PCR is only truly quantitative when measurements are taken
during the exponential phase, when reagents are not limiting and
the amount of PCR product is proportional to the quantity of the
input template. As the number of PCR cycles increases, the
reaction enters the plateau phase, where reagents become
limiting, the DNA polymerase loses activity, inhibitors build up
and the PCR product can accumulate to a concentration where
reannealing of the denatured DNA strands competes with primer
annealing. The cycle number at which the reaction transitions
from the exponential phase to the plateau phase varies with the
template, primers and reaction conditions. Real-time PCR allows
measurements to be taken throughout each cycle within the
exponential phase, improving accuracy and eliminating the time
and effort required for downstream analysis. The earliest real-
time PCR experiments involved