Applied Biosystems 7900HT User Manual

DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Appendix D Theory of Operation
D-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
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 Figure D-4 on page D-5 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 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
-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:
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.
Calculating
Threshold Cycles
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
T
) for a given amplification curve occurs at the point that the
fluorescent signal grows beyond the value of the threshold setting. The C
T
represents
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
T
s
To determine the C
T
for an Amplification plot, the SDS software uses data collected
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
n
values to generate a baseline
subtracted Amplification plot of ∆R
n
versus cycle number. Next, an algorithm
searches for the point on the Amplification plot at which the ∆R
n
value crosses the
threshold setting (the default threshold setting is 0.2). The fractional cycle at which
the intersection occurs is defined as the threshold cycle (C
T
) for the plot.
Note: It may be necessary to adjust the baseline and threshold settings to obtain
accurate and precise data. For further information on resetting the baseline and
threshold settings, see “Setting the Baseline and Threshold Values” on page 6-46.
N
c
N1 E+()
c
=

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English