Environmental Engineering Reference
In-Depth Information
to the template nucleic acid between the primers. As the nucleic acid polymerase
extends the primer, the probe is displaced, and the polymerase cleaves the fluo-
rogenic dye. Released dye is freed from the quencher and a fluorescent signal is
produced. The detection device consists of a multiwell thermal cycler connected
to a laser and a charge-coupled optics system. A fiber optic inserted through a
lens is positioned over each of the wells, and a laser beam is directed through
the fiber to excite fluorochrome in the PCR fluid present in wells. Fluorescence
emissions are sent through the fiber to the CCD camera, mathematically analyzed
by the system software, and the data are computerized.
Obtaining quantitative data on the original sample requires construction of a
calibration curve. This is done by preparing dilutions of a known quantity of
nucleic acid and performing PCR. Emissions data are obtained for each dilution
of the nucleic acid and plotted against thermal cycle numbers. A series of curves
result, and a line is drawn through the curves parallel to the thermal cycle numbers
(
x
axis) at a height just above the background fluorescence (Figure 1.8). Another
line is drawn perpendicular to the thermal cycles (
x
axis) at the intersection of the
parallel line and each of the curves representing the nucleic acid dilutions. The
thermal cycle number corresponding to each curve is the threshold cycle (
C
t). The
calibration curve is a plot of each
C
t value against the corresponding nucleic acid
concentration in the dilution series. The
C
t is inversely proportional to the copy
number (concentration) of nucleic acids in the dilution series, so a straight line
should result. The actual concentration of nucleic acid in the unknown sample is
determined by obtaining a
C
t value under identical conditions of PCR operation
10
1
10
0
10
−
1
10
−
2
C
t
02468 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0
Cycle
FIGURE 1.8
Family of fluorescence emission curves prepared from dilutions of nucleic
acid for the determination of threshold cycle values. (
Source
:
D. S. Grove, ”Quantita-
tive Real-Time Polymerasae Chain Reaction for the Core Facility Using TaqMan and the
Perkin-Elmer/Applied Biosystems Division 7700 Sequence Detector,” J. Biomol. Tech, 10
(1999): 11-16.
)
