Molecular biology: Real-time PCR
Author: Dr Kgomotso Sibeko-Matjilar
Licensed under a Creative Commons Attribution license.
Polymerase chain reaction (PCR) and DNA probebased techniques have improved the sensitivity and
specificity that previous
diagnostic
tests
lacked.
However, PCR and probing assays are relatively timeconsuming and labour intensive, particularly when
separate hybridization steps are required to confirm
test outcomes.
Recently, real-time PCR
improved
molecular
technology has greatly
detection
of
organisms
of
veterinary, medical and economic importance. The
real-time PCR technology employs similar amplification
processes
followed
in
traditional
PCR
methods
(denaturing, annealing and extension) however, the
real-time
PCR
technology
allows
fast
SYBR Green reaction
real-time
monitoring of a PCR reaction. Amplification and
accurate detection of specific targeted nucleic acid
sequences is accomplished in a single closed tube,
without the need for post-amplification analysis.
The real-time PCR
technology uses
fluorescent
detection systems to detect development of amplicons
during the amplification reaction. Therefore, the data is
measured at the exponential phase of the PCR
reaction rather than at the end-point of PCR as is the
case with the use of agarose gel electrophoresis. The
fluorescent detection systems employed in real-time
PCR reactions can be divided into two groups including
double stranded DNA intercalators, of which SYBR®
Green is widely used and single stranded detection
chemistries which are primer- or probe-based.
TaqMan® probe reaction
The double-stranded DNA intercalators bind on doublestranded DNA to detect PCR product as it accumulates
during PCR cycles. The single stranded detection
chemistry uses a fluorogenic probe to enable the
detection of a specific PCR product as it accumulates
during PCR cycles. The most important difference
between the two detection systems is that the SYBR
Green I dye chemistry will detect all double-stranded
DNA, including non-specific reaction products whereas
probe-based chemistries are more specific. A welloptimized reaction is therefore essential for accurate
results.
Advantages of using real-time PCR include:
 Collection of data occurs in the exponential
development phase (during the reaction) resulting in
high sensitivity.
 An increase in reporter fluorescent signal is directly
proportional to the number of amplicons generated.
 Differentiation between PCR products of the same
size
without
significant
sequence
variations
resulting in high specific detection.
 No requirement for post PCR processing, therefore
results are obtained faster when compared with
other tests.
 Contamination problems are significantly minimized
because of the closed-tube system.
 These qualities make real-time PCR assays ideal
for the diagnosis of infectious and parasitic
diseases, especially when pathogens are present at
very low levels.