Techne PrimeQ - Manual (Page 43)

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PCR (qPCR) extends the usefulness of the technology by permitting the reliable determination of
starting DNA template.
2.2.3 Real-time PCR on PrimeQ
Real-time PCR overcomes the limitations associated with the traditional methods by using
fluorescence labelling in conjunction with specialized amplification and detection systems to
quantify the amount of product being amplified during the PCR process. The fact that downstream
processing is eliminated saves both time and money while reducing the risk of contamination.
Further savings are to be found with the need for reduced replicates and that multiple reactions
can be combined within the same tube using different reporter dyes. However, the most notable
advantage over conventional approaches has to be its superiority in terms of accuracy and
sensitivity. As a result, this method has become a widely useful tool for the quantification of
messenger RNA or DNA levels in a wide range of biological samples.
PrimeQ provides a flexible approach to experiment setup by supporting real-time experiments for
monitoring the accumulation of PCR products during thermal cycling as well as end-point assays.
• Quantitative PCR: The advantage of this approach is that amplification of the accumulated
product is measured in the early exponential phase of the reaction at a point when the
amplified product can accurately reflect the starting DNA or RNA levels. By the end of the
experiment, reagents could be limiting which may mean that small differences in the PCR
performance are magnified. Additional benefits of this approach are its high sensitivity and
wide dynamic range. PrimeQ can measure to a sensitivity level of 1.0nM fluorescein and
with a dynamic range of at least nine orders of magnitude.
• End-point: This approach detects the presence or absence of an amplified product,
providing a qualitative positive or negative result. This approach can bring added flexibility to
the laboratory, allowing samples that have been run in a different thermal cycler to be
quickly screened for the presence of a PCR product by using PrimeQ as a plate reader.
In both approaches, the amount of product present in each reaction tube is visualized using
fluorescent reporter dyes.
2.3 Overview of fluorescent chemistries
An extremely sensitive system is required for the accurate visualization of small amounts of PCR
products. The conventional approach has been to use radioactivity, which although offering good
sensitivity, has obvious disadvantages in terms of safety and environmental risk. Fluorescence has
now taken over as the chemistry of choice due to its sensitivity, safety and the excellent flexibility it
lends to experimental setup.
Fluorescence is a molecular phenomenon in which a substance absorbs energy in the form of
light, causing it to first become
excited and then to emit part of this absorbed energy as light of
another colour. This light will be of a lower energy and therefore a longer wavelength (λ) of that
absorbed.
Schematic representation
of fluorescence.
When considering which fluorescent chemistry to use on PrimeQ, there are two main options:
fluorescence-labelled probes that bind specifically to the target of interest or intercalating dyes that
bind between the two strands of double-stranded DNA. The fluorescence signal seen when the