ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Fluorescence vs.
Amplified Product
When using TaqMan fluorogenic probes with the 7900HT instrument, fluorescence
emission increases in direct proportion to the amount of specific amplified product. As
the figure on page A-8 demonstrates, the graph of normalized reporter (R n ) vs. cycle
number during PCR appears to have three stages. Initially, R n appears as a flat line
because the fluorescent signal is below the detection limit of the Sequence Detector.
In the second stage, the signal can be detected as it continues to increase in direct
proportion to the increase in the products of PCR. As PCR product continues to
increase, the ratio of AmpliTaq Gold polymerase to PCR product decreases. When
template concentration reaches 10 -8 M, PCR product ceases to grow exponentially.
This signals the third stage of R n change, which is roughly linear and finally reaches a
plateau at about 10 -7 M (Martens and Naes, 1989).
The progressive cleavage of TaqMan fluorescent probes during the PCR makes
possible the correlation between initial template concentration and the rise in
fluorescence. As the concentration of amplified product increases in a sample, so
does the R n value. During the exponential growth stage (the geometric phase), the
relationship of amplified PCR product to initial template can be shown in the following
equation:
N
c
=
N1 ( + E)
c
where N c is the concentration of amplified product at any cycle, N is the initial
concentration of target template, E is the efficiency of the system, and c is the cycle
number.
Theory of Operation A-9