A New Tool for the Future
By
Lecturer of Virology, Faculty
of Veterinary Medicine, Cairo
University
12211, Giza-Egypt
Introduction:
The advent of
nucleic acid amplification and detection has resulted in a change from conventional
laboratory methods that rely on phenotypic characterization of pathogens and
antigens, to molecular techniques that enable more sensitive and rapid
identification of infectious agents. Real-time PCR is developed at 1992 as an
evolution of the basic PCR technique developed by Millus et al., and gained
wide acceptance due to its improved rapidity, sensitivity, specificity and the
reduced risk of crossover contamination. The principle of real-time PCR is
based principally on continuous detection of the amplified PCR product while the
amplification progress. This approach provides a great deal of insight into the
kinetics of the reaction and enables quantification of the target sequence in
the tested samples.
Real-Time
versus End-Point PCR:
Real-time PCR has a multitude of technical
advantages that render it superior to conventional end-point PCR. These
advantages include:
A)
Sensitivity
Real-Time PCR is the most sensitive molecular application method on the
market of molecular biology. Using its unique chemistries, it is now possible
to detect less copy numbers than with conventional PCR. Highly optimized
real-time PCR systems can detect down to a single copy of the target sequence
and can distinguish between few copies in the tested sample.
B)
Quantification
While the results obtained from conventional PCR is qualitative,
real-time PCR offers quantitative data. Qualitative results only give an
indication if the sample is positive or negative. The advantage of Real-Time
PCR is that quantitative analysis could be performed and copy numbers or microbial
load could be determined.
C)
Speed
Real-Time PCR is considered to be one of the fastest detection methods
in the field of diagnostic molecular biology. Conventional PCR usually takes up
to one day period to get final results. This delay is basically regarding to
the presence of three distinct steps of reaction: Nucleic acid extraction, PCR
cycling and post PCR analysis. With Real-Time amplification, time is obviously
reduced by omitting the post PCR step and decreasing the cycling protocol
duration, a matter that allows data collection and analysis within one to few
hours.
D)
Precision:
Real-Time PCR is nearly automated technology that requires very little
human interference. All the data are collected and analyzed by computer
software. This feature usually diminishes the human errors commonly experienced
in the conventional PCR as a consequence of visual interpretation of results
due to the lower gel resolution and size based discrimination of the amplified
fragments.
Since in Real-Time PCR the results are obtained during the run or
shortly after the run has finished, there is no need to open PCR tubes
containing amplified product. The risk of contaminating the work environment is
therefore strongly reduced.
F)
Minimizing Workload:
Real-Time PCR is much simpler than conventional PCR and requires less
experience, workload time and effort from the laboratory technician. In the traditional PCR, the product has to be
run on agarose gel after the cycling process has finish to verify the results,
while in Real-Time PCR, results are obtained immediately after the run or even
earlier while the assay is still running.
G)
Minimizing Workspace:
Space and construction are the major problems encounter designing special
molecular biology laboratories. In traditional PCR, three to four separate
rooms equipped with workstation cabinet, PCR thermal cycler, electrophoresis
chambers, power supplies, gel documentation system and some other accessories
should be available. These facilities require adequate space and special
constructions, which is not necessary in real-time PCR, where only a single or
double room containing a workstation cabinet, the Real-time PCR instrument and
some accessories are required.
H)
Mutation analysis
Traditionally mutation analysis is performed by conventional PCR
followed by a digestion with an appropriate restriction enzyme. In many cases,
known mutation cannot be analysis by the above method, since the lack of a
restriction enzyme side in this area. As a consequence, the PCR product needs
to be sequenced. Mutation analysis using Real-Time Amplification is able to
detect any known mutation using allelic discrimination assays or FRET assays.
I)
Higher throughput:
The number of samples that could be tested by
conventional PCR is limited by the practice of electrophoresis. A maximum of 20
or 30 samples could be tested at one shot in most cases. In real-time PCR, the
high speed data collection and automatic analysis of results enabled detection
of the full-capacity of the instrument at a time (up to 100 samples).
J)
Less Danger:
The use of highly carcinogenic substances like Ethidium bromide,
SYBRgreen,...etc in staining of the agarose gel is a necessary step for
visualizing the PCR product and getting results in end-point PCR. As a result
of removal of the post PCR analysis in real-time PCR, there is no need for
exposure to these dangerous substances that considered a potentional health
hazard.
2.
Real-Time PCR bases:
A) PCR phases:
In
order to understand the limitation of end point PCR and how real-time PCR was
developed, it is essential to outline the phases of PCR run. A standard PCR run
is composed of 25- 40 cycle, these cycles can be broken into three distinct
phases:
1- Exponential phase: Exact
doubling of product is accumulating at every cycle. The reaction is highly
specific and precise (reaction efficiency nearly 100%).
2- Linear (High variability) Phase: The
reaction components begin to consume. The reaction is slowing down and products
are starting to degrade (reaction efficiency less than 100%).
3- Plateau (End point): The
reaction has stopped with no more products accumulated. If left for a long
period, the PCR products will begin to degrade. At this phase, the products are
analyzed in agarose gel for conventional PCR.
B) Real-time PCR principle:
The real-time PCR is basically relied on
the same principle adopted for PCR amplification of specific DNA fragments. It
also needs the inclusion of a fluorescent reporter that binds either
specifically or non-specifically to the amplification product and generates a
fluorescent signal. The amount of emitted fluorescence is proportional to the
amount of the PCR product accumulated. During the initial cycles the signal is
week and can not be distinguished from the background. As the amount of product
accumulates, the signal increases exponentially and a responsive curve
develops. This curve is used to define
for calculation of the initial copy number of the target gene or sequence in
the sample at the beginning of reaction.
C)
Real-time PCR chemistries:
Four major chemistries are currently
in use for real-time PCR detection. They include:
1-
Intercalating dye:
Intercalating dyes, such as SYBR-Green I, RiboGreen, BEBO, YOYO, ....etc
bind to double stranded DNA.
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A
SYBR-Green (S) does not
bind to single stranded DNA and the intensity of fluorescent signal when S
excited by (E) is low.
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B
SYBR-Green (S) binds to double stranded DNA
and the intensity of fluorescent signal when S excited by (E) increases.
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SYBR-Green is a fluorescent
dye that binds to the minor groove of DNA double helix. The unbound dye
exhibits little fluorescence in solution, but upon binding to double-stranded
DNA the fluorescence is enhanced. This is utilized in real-time amplification.
As DNA is amplified during an amplification reaction, the dye binds to the
amplified product and the fluorescent signal is increased cycle after cycle.
Intercalating dyes can simply
be added to the amplification reaction tubes along with all the other
traditional amplification components: water, buffer, MgCl2, dNTP’s,
Taq-Polymerase, primers and template. This is an easy and cost effective
approach to real-time detection, as it does not require the design of sequence
specific probes and new primer sets.
Intercalating dyes are not sequence specific and
bind to any dsDNA including non-specific products and primer dimers. Therefore,
it is necessary to differentiate between target and artifact signals.
Intercalating dyes allow for the melting of amplification products at the end
of a run. This is called the melt curve analysis. During the melt curve, the
real-time machine continuously monitors the fluorescence of each sample as it
is slowly heated from a temperature below the melting point of the products to
a temperature above the melting point of the products. Amplification products
will melt at different temperatures based on their lengths and G/C content. As
products melt, a decrease in fluorescence is realized and measured by the
instrument. By taking the differential of the melt curve, the melting peaks can
be calculated. The melting peaks reflect the products amplified during the
reaction. These peaks are analogous to the bands on an electrophoresis gel.
B) Dual-Labeled Probes (Hydrolysis or TaqMan
Probes):
Dual labeled probes are short oligos with a
fluorescent reporting dye attached to the 5’-end and a quencher molecule
attached to the 3’-end. Because the probes are only 15-25 bp long, the reporter
dye and quencher are in close proximity to each other and little fluorescence
is detected. During the cycling process, Taq DNA-polymerase extends from each
primer. The DNA polymerase has an exonuclease activity that cleaves the
downstream probe as it extends. As the probe is degraded, the reporter dye is
separated from the quencher.
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A
Energy emitted by the
donor fluorophore (D) when excited by (E) is absorbed by the nearby quencher
(A).
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B
The polymerase exonuclease activity separates the
fluorophore donor (D) from the quencher (A) by hydrolysis and resulting in an
increase in fluorescent signal when D excited by E.
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With every cycle of amplification an increase in
reporting dye is detected by the real-time instrument due to the cleavage of
the probes. Because the reporting dye is cleaved, melt curve analysis is not
possible. Dual labelled probes offer higher specificity and sensitivity compared
to intercalating dyes. There are two reasons for this. First, dual labelled
probes are sequence specific and only bind to complimentary regions. The second
reason is that the dual labelled probe for each amplified copy releases only
one molecule of fluorescent dye.
C) FRET Probe System (Hybridization or
LightCycler Probes):
FRET probes rely on the transfer of energy from
one fluorescent dye to another. Two separate sequence specific oligos are
fluorescently labelled, one molecule (donor) on the 3’-end and the other (acceptor)
on the 5’-end. The probes are designed so that they hybridise adjacently to
each other on the target sequence and bring the donor and acceptor fluorophores
in close proximity. This allows transfer of energy from the donor to the
acceptor fluorophore, which emits a signal of a different wavelength. Either
the decrease in the fluorescence of the donor or the increase in fluorescence
of the acceptor can then be detected. Therefore, only when both probes are
bound is fluorescence detectable.
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A
Energy emitted from the
donor after excitation by (E) is low when the probes are not hybridized.
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B
Hybridization of the
probes brings the donor (D) and acceptor (A) fluorophores into close
proximity resulting in increased energy transfer and fluorescence emitted
from the acceptor.
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3. Applications of Real-Time:
A.
Pathogen detection
and quantitation either virus, bacteria, protozoa, fungi, algae,…etc.
B.
Genotypic
of different pathogenic organism.
C.
Quantitative
analysis of gene expression in immunological studies, cancer research and drug
therapy efficacy.
D.
Mutation
detection and analysis of different aspects of genetic diversity.
E.
Quality
control of different biological materials and Assays validation.
References:
1- Kubista, M.; Andrade, J.;
Bengtsson, M.; Forootan, A. et al., (2006). The real time polymerase chain reaction.
Molecular Aspects of Medicine, 27: 95-125.
2- Mackay, I.
M. (2004). Real-time PCR in the microbiology laboratory. European Society of
Clinical Microbiology and Infectious Diseases.
3- Klein, D. (2002).
Quantification using real-time PCR technology: application and limitation.
TRENDS in Molecular Medicine, 8 (6): 257-260.
4- Mackay, I.; Arden, K. E. and
Nitsche, A. (2002). Real-time PCR in Virology. Nucelic Acids Research, 30
(6): 1292-1305.
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