The present invention concerns methods of quantifying nucleic acids using
a bioluminescence regenerative cycle (BRC). In BRC, steady state levels
of bioluminescence result from processes that produce pyrophosphate.
Pyrophosphate reacts with APS in the presence of ATP sulfurylase to
produce ATP. The ATP reacts with luciferin in a luciferase-catalyzed
reaction, producing light and regenerating pyrophosphate. The
pyrophosphate is recycled to produce ATP and the regenerative cycle
continues. Because the kinetic properties of ATP sulfurylase are much
faster than luciferase, a steady state results wherein concentrations of
ATP and pyrophosphate and the rate of light production remain relatively
constant. Photons are counted over a time interval to determine the
number of target molecules present in the initial sample. The BRC process
has a controllable dynamic range up to seven orders of magnitude and is
sensitive enough to detect a few thousand molecules of target nucleic
acid.