Biomedical Engineering Reference
In-Depth Information
1.2. The Principle of Relative Quantification by Real-Time PCR
in Relation to the Determination of Chromosome Ploidy
Real-time PCR is based on the detection and quantitation of a fluorescent
signal that is directly proportional to the PCR product being generated during
each cycle of the PCR. In our assay, the fluorescence is generated by the 5'
nuclease method: a so-called TaqMan probe hybridizes specifically to one
strand of the DNA sequence between the two primers. The probe is labeled
with a fluorescent detector dye at its 5' end and with a quencher dye at its 3'
end. The annealing temperature of the probe is 10°C higher than the annealing
temperature (T
A
) of the PCR reaction, which is the melting temperature of the
primers. This ensures that in each cycle of the reaction, a probe binds to every
target sequence before the primers anneal. When the primer binds to the target,
the
Taq
polymerase immediately starts extending the primer and by its nuclease
activity cleaves the 5' end of the probe. This nucleolytic activity separates the
detector dye from the quencher dye, thereby permitting the detector dye to
emit a characteristic fluorescence signal when excited by an appropriate light
source. As the displacement of each probe molecule is the result of a single
template amplification, the amount of fluorescent signal measured is therefore
directly proportional to the number of probes cleaved and the amount of PCR
product synthesized.
For the correct analysis of such real-time PCR assays, several parameters
have been devised. The first of these is the C
T
-value, or threshold cycle, defined
as the cycle number at which point the amplification curve crosses the thresh-
old line in semi-log view of the amplification plot (
Fig. 1
). Although the
C
T
-value is the chief factor used in most real-time PCR analyses, in our experi-
ence another important parameter, especially for the analysis of discrete tem-
plate increments, is the normalized final fluorescence,
R
n
-value, a
measure of the accumulation of specific fluorescence, is indicative of the am-
plification efficiency and initial template concentration (
Fig. 1
).
As our real-time PCR test for fetal aneuploidy entails the simultaneous am-
plification of two chromosomal loci (e.g., chromosome 21 vs chromosome 12
or chromosome 21 vs chromosome 18) in a multiplex reaction, their product
formation is detected by two different fluorescent dyes. As each of these re-
porter dyes has a discretely different emission spectrum, and as the amount of
each dye measured is proportional to the relevant target template, this enables
the relative quantification of both chromosomes. This can be calculated by
using the
∆
R
n
. This
∆
C
T
, which is the difference between the C
T
-values of the first (e.g.,
chromosome 18, VIC dye) and the second (e.g. chromosome 21, FAM dye)
amplified sequences in one reaction well (
Fig. 2
):
∆
∆
C
T
= C
T
(target A) - C
T
(target B)
= C
T
(VIC, Chromosome 18) - C
T
(FAM, Chromosome 21)
