The PCR Blog: The Polymerase Chain Reaction

History of Real-time PCR

Originally, the simultaneous amplification and detection of specific DNA sequences in real-time was achieved by adding ethidium bromide (EtBr) to the PCR reaction so that the accumulation of PCR product could be visualized at each cycle. When EtBr is bound to double-stranded DNA and excited by UV light it fluoresces, therefore an increase in fluorescence in the reaction indicates positive amplification. Soon afterwards real-time PCR product quantitation or "kinetic PCR" was achieved by continuously measuring the increase in EtBr intensity during amplification with a charge-coupled device camera. By creating amplification plots of fluorescence increase versus the cycle number the kinetics of EtBr fluorescence accumulation during thermocycling was directly related to the starting number of DNA copies. When a greater number of target molecules are present fewer cycles are needed to produce a detectable signal.

Kinetic monitoring also provided a means whereby the efficiency of amplification under different conditions could be determined, providing for the first time insight into the fundamental PCR processes. The principle underlying quantitative real-time PCR can be defined as the monitoring of fluorescent signal from one or more PCR reactions, cycle-by-cycle, to completion, where the amount of product produced during the exponential amplification phase can be used to determine the amount of starting material.

The use of EtBr was not ideal since EtBr binds non-specifically to DNA duplexes and non-specific amplification products, such as primer–dimers, can contribute to the fluorescent signal and result in quantification inaccuracies. Subsequent refinements, the most significant of which was the introduction of fluorogenic probes to monitor product accumulation, added a greater element of specificity to real-time PCR and provided greater quantitative precision and dynamic range than previous methods.

These significant advances to the basic PCR technique led to the development of a new generation of PCR platforms and reagents, which allowed simultaneous amplification and quantification of specific nucleic acid sequences cycle-by-cycle. The first commercial platform to become available was the Applied Biosystems ABI Prism 7700 Sequence Detection System, followed by the Idaho Technology LightCycler (later manufactured and sold by Roche Diagnostics). Both of these platforms utilized fluorogenic chemistry and like any real-time PCR platform, they basically consist of a thermal cycler with an integrated optical detection system, which can heat, cool, detect and report. New and improved models have now superseded these two instruments and several other manufacturers have introduced their own real-time PCR platforms.

Real-time PCR offers many advantages over traditional PCR, including the amplification and detection in an integrated system, fluorescent dyes/probes allowing constant reaction monitoring, rapid cycling times (20-40 mins for 35 cycles), high sample throughput (200 to 5000 samples/day), low contamination risk due to sealed reactions, increased sensitivity, detection across a broad dynamic range of 10 - 1010 copies, reproducibility, quantification of results, and software driven operation.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Labels: ethidium bromide, qPCR, quantitation, quantitative real time pcr, real-time pcr, real-time PCR history

History of Real-time PCR

Real-Time PCR Data Analysis

Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

The software provided with real-time PCR instruments allows several types of data analysis:

normalisation of the raw data
measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
melting curves are transformed to provide plots of –dF/dT against T (F = fluorescence and T= temperature) in which a peak (melting peak) occurs at the equilibrium temperature for each duplex

In general instrument specific software is easy to use and allows rapid and reproducible data analysis. In addition to the bundled software a range of third party utilities is available to improve the flexibility of real-time PCR data analyses.

from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR

Labels: data analysis, PCR instruments, qPCR, qRT-PCR, quantitation, quantitative real time pcr

Real-Time PCR Data Analysis

PCR Quantification

Unlike standard PCR, real-time PCR instruments measure the kinetics of product accumulation in each PCR reaction tube. Generally, no product is detected during the first few PCR cycles as the fluorescent signal is below the detection threshold of the instrument.

Most combinations of machine and fluorescence reporter are capable of detecting the accumulation of amplicons before the end of the exponential amplification phase. During this time the efficiency of PCR is often close to 100% giving a doubling of the quantity of product at each cycle. As product concentrations approach the nanogram per microlitre level the efficiency of amplification falls primarily because the amplicons re-associate during the annealing step. This leads to a phase during which the accumulation of product is approximately linear with a constant level of net synthesis at each cycle. Finally, a plateau is reached when net synthesis approximates zero.

Quantification in real-time PCR is done by measuring the number of cycles required for the fluorescent signal to reach a threshold level or the second derivative maximum of the fluorescence versus cycle curve. This cycle number is proportional to the number of copies of template in the sample. Real-time PCR quantification applications are discussed in detail in Bustin and Nolan (2009) Analysis of mRNA Expression by Real-Time PCR In: Real-Time PCR Logan, Edwards and Saunders, eds.; Wurmbach (2009) Validation of Array DataIn: Real-Time PCR Logan, Edwards and Saunders, eds.; and Wiseman (2009) Real-Time PCR: Application to Food Authenticity and Legislation In: Real-Time PCR Logan, Edwards and Saunders, eds.

Bibliography:

Labels: fluorescence, PCR instruments, PCR machines, quantitation, real-time pcr

PCR Quantification

Real-time PCR: Advantages over standard PCR

Standard PCR requires the identification of the amplified fragment(s) by post-PCR analysis, usually by gel electrophoresis. These methods rely on either the size or sequence of the amplicon. Gel electrophoresis, often used to measure the amplicon size, is both inexpensive and simple to implement. However, size analysis has limited specificity since different molecules of approximately the same molecular weight cannot be distinguished. Gel electrophoresis alone is not a sufficient PCR end-point in many instances, including most clinical applications. Characterisation of the product by its sequence is far more reliable and informative and probe hybridisation assays can be used for this purpose. Such methods are time-consuming and care must be taken to ensure that amplicons accidentally released into the laboratory environment do not contaminate the DNA preparation and clean rooms.

Real-time PCR greatly simplifys amplicon recognition by providing the means to monitor the accumulation of specific products continuously during cycling. All current instruments designed for real-time PCR, measure the progress of amplification by monitoring changes in fluorescence within the PCR reaction vessel. Changes in fluorescence can be linked to product accumulation by a variety of methods.

A further advantage of the real-time format is that the analysis can be performed without opening the tube which can then be disposed of without the risk of dissemination of PCR amplicons or other target molecules into the laboratory environment. Although alternative methods for avoiding PCR contamination are available, containment within the PCR vessel is likely to be the most efficient and cost-effective.

A major drawback of standard PCR formats that rely on end-point analysis is that they are not quantitative because the final yield of product is not primarily dependent upon the concentration of the target sequence in the sample. Real-time PCR overcomes this limitation.

from N.A. Saunders in Real-Time PCR: Current Technology and Applications

Bibliography:

Labels: contamination, quantitation, real-time pcr

Real-time PCR: Advantages over standard PCR