Real time PCR - European Pharmaceutical Review

qPCR ISSUE 2009
assay) and a melting curve analysis
(see Figure 8 on page 12) have also
been developed, which are more
qualitative. Both assay allow the
detection of a specific sequence
(i.e. genetic polymorphism) rather
Figure 6: These two plots illustrate the high sensitivity of real time PCR. On the left, plots
show fluorescence traces obtained over 6 log dilution of the same sample and on the
right, the linearity over the detection rage. (Adapted from: Real-Time PCR. Chapter 8:
Real-time PCR using SYBR® Green I.
By Dr Frederique Ponchel. Talyor &Francis. 2006 Oxford Press)
than their quantification, however,
this has also been done to calculate
the expression of specific alleles.
This particular real-time PCR
application has contributed to the
development of high throughput
allelic discrimination assays.
Since the original release of the
first real time PCR machines by ABI in
the late 90s, a number of platforms
have been developed by several
companies (Biorad, Roche, Hybaid,
Eppendorf …). Similarly, there are now
many commercially available TaqMan
“off the shelf” assays designed by
companies, several real time grade
Taq polymerase, several TaqMann
chemistry kits or SYBR-green kits
(ABI, Quiagen, Sigma, Roche, Biorad,
BioScience corp., MJ Research and
probably many others). Today, there
are no reason to chose one over the
other however, once an assay has
been optimised with one chemistry,
changing kit requires re-optimisation.
Multiple software for designing
primers and probes have been
developed due to the increasing
popularity of the technology.
Accordingly, the risk that real-time
PCR technology may be used
inappropriately or at least without
proper optimisation and
understanding of its limitations, has
raised considerably.
Applications using the real
time PCR technology
Figure 7: In this assay, the same forward and reverse primers, are used to generate a PCR
reaction. Two Taqman” probes are use in the same assay however each of them is
specific for one allele of the gene, with either a perfect hybridisation on one allele but
allowing the presence of a mismatch on the second allele. When the polymerase
approaches, the probes are lifted from the template. If the strength of the hybridisation is
sufficient to maintain the probe on the template the 5’ exonuclease activity cleaves the
probe and releases fluorescence. In presence of the mismatch, the probe is getting away
before the exonuclease activity can cleave it therefore preventing the release of the
fluorescent dye. (Adapted from: ABI web site).The ratio of fluorescence for each allele
(bottom graph,) is then calculated at the end of the assay and either biased to one or the
other allele (homozygotes: allele 1 red, allele 2 blue, negative control black) or mixed
(heterozygote, green).
Quantification of genomic DNA
Medical diagnostic or prognostic is
one of the areas where real time
PCR has really made a difference.
There are several situations where
quantifying DNA is important.
The short response time to obtain
an outcome can often be of great
importance for patients, particularly
when considering that the alternative
methods may sometimes require
live cells or long delays. Gene
rearrangements or translocations
have been studied using real-time
PCR. For example, the major and
minor break-points of the
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