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الثلاثاء، 31 يناير، 2012

Real-time PCR



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.


A
SYBR-Green (S) does not bind to single stranded DNA and the intensity of fluorescent signal when S excited by (E) is low.

B
 SYBR-Green (S) binds to double stranded DNA and the intensity of fluorescent signal when S excited by (E) increases.



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.




A
Energy emitted by the donor fluorophore (D) when excited by (E) is absorbed by the nearby quencher (A).

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.


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.


A
Energy emitted from the donor after excitation by (E) is low when the probes are not hybridized.

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.



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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