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
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: ethidium bromide, qPCR, quantitation, quantitative real time pcr, real-time pcr, real-time PCR history
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