Azure Cielo Real-Time PCR Systems User Manual
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4. Frequently Asked Questions
General questions
How is qPCR different than regular PCR?
Quantitative PCR, using fluorescent detection chemistry, can determine starting quantities of materials if
performed correctly with all necessary controls. It can be monitored as the run progresses with the fluorescent
signal measured and recorded during the exponential phase of PCR, when the reaction is at its highest
efficiency. Conventional PCR is evaluated after the run is complete (also known as End Point detection)
and after which reaction components become limited at different rates and slow the reaction. As a result,
accurate quantitation is not possible.
What is a threshold cycle?
A threshold cycle (C
t
) or quantification cycle (C
q
) is the cycle at which the fluorescent detection chemistry
reaches a level above background. It is determined by the instrument software and is inversely proportional
to the concentration of the target. If there are many copies of the target sequence present, it will reach the
threshold level earlier in the run and have a lower C
t
/C
q
. If there is a low amount of target, it will take longer
for the cycling to achieve that threshold level and will have a later (or higher) C
t
/C
q
.
What is the difference between SYBR green and probe-based qPCR? Why would I use one over the other?
SYBR Green detection chemistry is a nonspecific, double-stranded binding dye. Unbound, it has a very low
fluorescent signal, but once attached to double-stranded DNA, it emits brightly and is easily detected. The
nonspecific nature of SYBR Green (and other nonspecific binding dyes) means it will bind to any double-
stranded DNA fragments in an amplification reaction, including nonspecific targets such as primer dimers. By
running a melt or dissociation curve at the completion of the run, specificity of the fluorescent signal can be
determined with a high level of confidence.
The most common probe-based chemistry is hydrolysis probes. A probe-based assay consists of two primers
along with a probe that will target a sequence located between the two primers. Probe structure does vary,
but generally consists of a fluorescent tag on one end of the probe, with a light quenching molecule on the
other end. When the probe is intact, the quenching molecule will absorb any signal from the fluorophore.
As PCR proceeds, the exonuclease activity of Taq DNA polymerase will cleave the probe. By this action, the
quenching molecule and the fluorescent dye molecules are now separated from one another, allowing a
robust fluorescent signal. With each cycle, more of the probe is cleaved in this process, until detectable signal
is achieved. Probe-based detection is highly sequence specific, so melt curve analysis is not necessary at the
end of the run.
There are many factors that should be considered when picking which detection chemistries to use. SYBR
Green is less expensive, and if many different primer sets are in use, might be the more flexible choice. Probe-
based has a higher cost but is also highly specific, and with the large variety of fluorophores available, allows
for multiplexing of assays.
What exactly is a negative control? Why do I need it?
A negative control for PCR is also known as a no template control. This is a reaction that contains all reaction
components, such as primers, dNTPs and Taq DNA Polymerase, but does not contain any template. It is
usually done in conventional PCR as well, with the main purpose to show lack of contamination during set up.