Techne PrimeQ - Manual (Page 105)

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3.9 Introduction
PrimeQ can be used to determine the absolute or relative quantity of a target DNA template in a
given test sample by measuring the cycle-to-cycle change in the fluorescent signal. It can also
measure the simple presence or absence of a target. The fluorescent signal is taken to increase
proportionately to the amount of amplified DNA and quantification is performed by comparing the
fluorescence of a PCR product of unknown concentration with the fluorescence of several dilutions
of an external standard. To be able to make this comparison, however, the fluorophore must be
measured at a point in the amplification where the reaction efficiency can be considered
comparable. This concept can be explained more clearly by looking at a typical amplification
curve.
3.9.1 Amplification curve
If a PCR is plotted as fluorescence against cycle number, amplification of a PCR product would
generate a curve similar to the one shown below.
The reaction curve is made up of distinct regions characteristic of PCR progression.
• Lag phase (background): Low-level fluorescence seen in the early cycles of the PCR while
the target DNA copy number is still low. This can be subtracted from the readings to
eliminate the level of background fluorescence. Background subtracting the data allows the
user to calculate the point at which the DNA has been amplified to a level that can be
detected above the noise.
• Growth phase (exponential): The most useful data occurs during the cycles where
readings are above the noise and increasing in an exponential fashion. The phase is
characterized by high and constant amplification efficiency and occurs between the first
detectable increase in fluorescence and the plateau phase. If fluorescence vs. cycle number
is plotted on a log scale, the plot should be a straight line.
• Linear phase: This phase is characterized by a steady levelling off of the amplification
curve. At this point, at least one of the reaction components has fallen below a critical level
and the amplification efficiency has begun to decrease (in a 100% efficient reaction, a
doubling of the target should occur every cycle). As the efficiency is steadily decreasing
during this phase, comparison between wells is likely to be less accurate.
• Plateau phase: As seen in a typical PCR curve, the linear phase eventually plateaus and
stabilizes. At this point, one or more of the components is limiting the reaction and the
increase in DNA amplification stops.
3.9.2 Thresholds
The threshold fluorescence is the point at which a reading is considered to be significantly above
the noise. At this point, the reaction efficiency is expected to be at its highest with no limiting
effects from reagent exhaustion.
Cycle Num ber
45 40 35 30 25 20 15 10 5
R
e
la
ti
v
e
F
lu
o
re
s
c
e
n
c
e
2.5
2
1.5
1
0.5
0
Lag phase
Growth phase
Linear phase
Plateau phase
Cycle Num ber
45 40 35 30 25 20 15 10 5
R
e
la
ti
v
e
F
lu
o
re
s
c
e
n
c
e
2.5
2
1.5
1
0.5
0
Lag phase
Growth phase
Linear phase
Plateau phase