Real time PCR - European Pharmaceutical Review

qPCR ISSUE 2009
Real time PCR
Dr. Frédérique Ponchel, Senior Research Fellow, Leeds Institute of Molecular Medicine, University of Leeds
Polymerase Chain Reaction (PCR) has revolutionised molecular biology, and is now a wellstandardised
procedure and its applications are wide. Real time PCR is a more recent development
of the initial PCR reaction, which has provided a solution to the limitation of standard PCR with
regards to quantification. By using fluorescence, which allows the quantification of PCR products at
each cycle of the reaction, real time PCR avoids the “plateau” effect of standard PCR. Two main
approaches have been developed, the TaqMAn assay and the SYBR-Green I dye. Alongside realtime
PCR as such, an “end point assay” has also evolved, most often referred to as allelic
discrimination, allowing the detection of a specific sequence (i.e. genetic polymorphism). The
technology is now widely used for DNA and RNA quantification as well as genetic polymorphism
detection. It can be applied to the detection and quantification of plain mRNA, for splice variant,
alternative promoter usage, allele specific expression etc. For DNA, gene amplification, gene
deletion or rearrangement, allelic copy number can be measured. It is also widely used for the
detection of pathogens or contaminants in food or other types of products.
Figure 1: PCR can amplify a small amount of template DNA (or RNA) into large quantities
in a few hours. This is performed by mixing the DNA with primers on either side of the
DNA (forward F and reverse R), Taq polymerase, free nucleotides (dNTPs), and buffer.
The temperature is then alternated between hot and cold to denature and reanneal the
DNA, with the polymerase adding new complementary strands each time.
Adapted from http://www.slideworld.org/viewslides.aspx/Real%20Time%20PCR%20principle%20%20chemistry%20%20application%20-%2066144
The advent of Polymerase Chain
Reaction (PCR) almost 30 years
ago has revolutionised
molecular biology. The principles of
PCR are simple but rely on a
particular type of DNA poplymerase,
the Taq polymerase (from Thermus
aquaticus). This form of the enzyme
is thermophile and its polymerase
activity is able to withstand
extremely high temperatures allowing
cycles of high and low temperature.
The PCR reaction amplifies a low copy
number nucleic acid template (DNA
or RNA) into detectable amount,
allowing its analysis (see Figure 1).
PCR is now a well-standardised
procedure and its application are wide.
Real time PCR is a more recent
development of the initial PCR
reaction and its popularity has never
ceased growing since the late 90s as
illustrated by the increasing numbers
of publication using this technique
(see Table 1 on page 11). It resolved
the main limitation of the standard
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