13
Basics of real-time PCR
1
For Research Use Only. Not for use in diagnostic procedures.
TaqMan
®
probe signal production
Whether an MGB or non-MGB probe is chosen, both follow
the same pattern for signal production. In the early PCR
cycles, only the low, quenched reporter signal is detected.
This early data, automatically subtracted to zero in the real-
time PCR software, is termed “baseline.” If the sample
contains target, eventually, enough accumulated cleaved
probe will be produced to allow amplification signal to
emerge from the baseline. The point at which amplification
signal becomes visible is inversely related to the initial
target quantity.
SYBR
®
Green dye
SYBR
®
Green I dye is a fluorescent DNA binding dye,
binding to the minor groove of any double-stranded DNA.
Excitation of DNA-bound SYBR
®
Green dye produces a
much stronger fluorescent signal compared to unbound
dye. A SYBR
®
Green dye–based assay typically consists
of two PCR primers. Under ideal conditions, a SYBR
®
Green assay follows a similar amplification pattern as a
TaqMan
®
probe-based assay. In the early PCR cycles, a
horizontal baseline is observed. If the target was present
in the sample, sufficient accumulated PCR product will
be produced at some point so that amplification signal
becomes visible.
SYBR
®
Green assay specificity
Assay specificity testing is important for all assays, but
especially for those most vulnerable to specificity problems.
SYBR
®
Green assays do not benefit from the specificity
of a TaqMan
®
probe, making them more vulnerable to
specificity problems. SYBR
®
Green dye will bind to any
amplified product, target or non-target, and all such
signals are summed, producing a single amplification plot.
SYBR
®
Green amplification plot shape cannot be used to
assess specificity. Plots usually have the same appearance,
whether the amplification consists of target, non-target, or
a mixture. The fact that a SYBR
®
Green assay produced an
amplification should not be automatically taken to mean
the majority of any of the signal is derived from target.
Since amplification of non-target can vary sample-to-
sample, at least one type of specificity assessment should
be performed for every SYBR
®
Green reaction. Most
commonly, this ongoing assessment is the dissociation
analysis.
SYBR
®
Green dissociation
SYBR
®
Green dissociation is the gradual melting of the PCR
products after PCR using SYBR
®
Green. Dissociation is an
attractive choice for specificity assessment because it does
not add cost to the experiment and can be done right in the
PCR reaction vessel. However, dissociation does add more
time to the thermal protocol, requires additional analysis
time, is somewhat subjective, and has limited resolution.
The concept of SYBR
®
Green dissociation is that if the
target is one defined genetic sequence, it should have one
specific melting temperature (T
m
), which is used to help
identify the target in samples. Some non-target products
will have T
m
s significantly different from that of the target,
allowing detection of those non-target amplifications.
The dissociation protocol is added after the final PCR cycle.
Following the melt process, the real-time PCR software
will plot the data as the negative first derivative, which
transforms the melt profile into a peak.
Accurate identification of the target peak depends on
amplification of pure target. Many samples such as cellular
RNA and genomic DNA exhibit high genetic complexity,
creating opportunities for non-target amplification that
may suppress the amplification of the target or, in some
cases, alter the shape of the melt peak. By starting with
pure target, the researcher will be able to associate a peak
T
m
and shape with the a particular target after amplification.
Only one peak should be observed. The presumptive target
peak should be narrow, symmetrical, and devoid of other
anomalies, such as shoulders, humps, or splits. These
anomalies are strong indications that multiple products
of similar T
m
s were produced, casting strong doubts about
the specificity of those reactions. Wells with dissociation
anomalies should be omitted from further analysis.
SYBR
®
Green dissociation is low resolution and may not
differentiate between target and nontarget with similar
T
m
s, e.g., homologs. Therefore, one, narrow symmetric
peak should not be assumed to be the target, nor one
product, without additional supporting information.
Dissociation data should be evaluated for each well where
amplification was observed. If the sample contains a
peak that does not correspond to the pure target peak,
the conclusion is that target was not detected in that
reaction. If the sample contains a peak that appears to
1.5 Real-time PCR fluorescence detection systems