Applied Biosystems 7900HT User Manual page 352

Appendix D Theory of Operation
Fluorescence vs.
Amplified
Product
Calculating
Threshold Cycles
D-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, App_Theory.fm
When using TaqMan fluorogenic probes with the 7900HT instrument, fluorescence
emission increases in direct proportion to the amount of specific amplified product.
As Figure D-4 on page D-5
cycle number during PCR appears to have three stages. Initially, R
line because the fluorescent signal is below the detection limit of the 7900HT
instrument. 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
exponentially. This signals the third stage of R
finally reaches a plateau at about 10
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 value. During the exponential growth stage (the geometric phase), the
n
relationship of amplified PCR product to initial template can be shown in the
following equation:
) c
(
N c = N 1 + E
where N is the concentration of amplified product at any cycle, N is the initial
c
concentration of target template, E is the efficiency of the system, and c is the cycle
number.
The Applied Biosystems 7900HT Fast Real-Time PCR System creates quantifiable
relationships between test samples based on the number of cycles elapsed before
achieving detectable levels of fluorescence. Test samples containing a greater initial
template number cross the detection threshold at a lower cycle than samples
containing lower initial template. The SDS software uses a Threshold setting to
define the level of detectable fluorescence.
The threshold cycle (C
fluorescent signal grows beyond the value of the threshold setting. The C
a detection threshold for the 7900HT instrument and is dependent on two factors:
• Starting template copy number
• Efficiency of DNA amplification the PCR system
How the SDS Software Determines C
To determine the C for an Amplification plot, the SDS software uses data collected
T
data from a predefined range of PCR cycles called the 'baseline' (the default
baseline occurs between cycles 3 and 15). First, the software calculates a
mathematical trend based on the baseline cycles' R
subtracted Amplification plot of ∆R
searches for the point on the Amplification plot at which the ∆R
threshold setting (the default threshold setting is 0.2). The fractional cycle at which
the intersection occurs is defined as the threshold cycle (C
It may be necessary to adjust the baseline and threshold settings to obtain
Note:
accurate and precise data. For further information on resetting the baseline and
threshold settings, see
DRAFT
demonstrates, the graph of normalized reporter (R
-7
M (Martens and Naes, 1989).
) for a given amplification curve occurs at the point that the
T
s
T
versus cycle number. Next, an algorithm
n
"Setting the Baseline and Threshold Values" on page
appears as a flat
n
-8
M, PCR product ceases to grow
change, which is roughly linear and
n
values to generate a baseline
n
value crosses the
n
) for the plot.
T
) vs.
n
represents
T
6-46.