Real-Time PCR

Monitoring DNA amplification

Real-time polymerase chain reaction (RT-PCR), also known as quantitative PCR, provides an elegant solution for quantification of DNA sequences in real time, as opposed to conventional PCR, where only the final result of the PCR amplification can be analyzed. The amplification reaction progresses due to repeated cycles of heating and cooling at predefined temperatures for every stage, which are referred to as denaturation, annealing and elongation. The PCR also requires specific reagents, namely DNA template, DNA polymerase, primers (oligonucleotides) serving as starting points and thereby guiding the amplification, as well as dNTPs, serving as building blocks. In order to follow the amplification process in real-time, fluorescent dyes are linked to the primers so that the DNA amplification can be traced; i.e. the fluorescent signals are effectively reporting snapshots of the reaction to the software. The reaction can either be traced with one dye only, e.g. SYBR® Green, or with up to 6 dyes for multiplex RT-PCRs where several DNA targets are amplified simultaneously. This requires a RT-PCR cycler with 6 optical channels, as in the Rotor-Gene Q. Throughout the amplification process, the RT-PCR cycler will send optical data to its software, which will display the results as a curve indicating the amount of signal, i.e. the amount of amplification, in each cycle.

Sensitivity and accuracy

Successful RT-PCR runs rely on high-precision technology; they require a reliable RT-PCR cycler with minimal tube-to-tube variation, fast ramp rate, and optical and thermal accuracy. Different solutions can be used to reach a precise and uniform temperature across all samples in a short period of time during the reaction; for example, Peltier elements and air flow or liquid flow are efficient in controlling and evening out the temperature. The excitation of the fluorescent dyes can be achieved by light sources, such as a halogen, UV, LED lights or a Xenon lamp in combination with dye-specific filters. The signals are captured by a detector (e.g. a photodiode or a CCD), amplified by the photomultiplier and sent to the software.

Downstream applications

Over the years, the technology mentioned above has enabled RT-PCR cyclers to become increasingly sophisticated and reliable, and hence today RT-PCR plays an essential role in many biomedical applications such as gene expression analysis, pathogen detection, DNA methylation analysis, SNP genotyping and gene scanning, as well as miRNA research.

For information about products based on this technology, see Rotor-Gene Q.

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