It was the beginning of a revolution in Molecular biology in 1983 when Kary Mullis first introduced the concept of in-vitro amplification of DNA with a polymerase enzyme. Since then, several advances and derivations of this method has lead to numerous applications in molecular biology.
Real time PCR (qrt-PCR – Quantitative Real Time PCR) is one such PCR based method that actually incorporates amplification and simultaneous quantification of amplicons. During conventional PCR, the product is detected at it’s end but with qrt-PCR the product is detected during the progress of the reaction. The really useful application of PCR relies on quantification of m-RNA, in which qrt-PCR is coupled to reverse transcriprase PCR.
The principles behind quantification
Two commonly used methods are available for the quantification procedure as every other process is the same as standard PCR. One method relies on fluorescent dye molecules. A fluorescent dye which has the ability to bind double stranded DNA is added to the usual reaction setup. As the DNA amplifies with each PCR cycle, the dye bound double stranded DNA increases which in turn causes an increase in fluorescence. The fluorescence intensity is measured at each cycle and the DNA is thus quantified. The major drawback of this method is the interference of non-specific ds-DNA products such as primer dimers. As these products are also ds-DNA, the fluorescent dye could bind to them as well.
The other method relies on a fluorescent probe. During the setup of the reaction mixture, the fluorescent reporter probes are added. During the annealing step the primers and the probes anneal. As Taq polymerase amplifies target DNA, the exonuclease activity of the enzyme removes the fluorescent probes bound to the target DNA. This causes an increase in fluorescence and subsequently detected by the real time PCR machine. This method is very specific as the probes anneal only perfectly complementary DNA. Specific characteristics of the probe enables fluorescent detection. This fluorescent probe is designed so that there is a fluoroscent reporter at one end and a quencher at the other end of the probe. The close proximity of the reporter to the quencher prevents fluorescent detection. When the exonuclease activity degrades the probe, quencher and reporter are separated so that fluorescence is unquenched and detectable. Thus fluorescence increases with increasing probe degradation.
qrt-PCR has enormous molecular biological applications. Mainly utilized in research and molecular disease diagnosis. As mentioned above, qrt-PCR is used in gene expression analysis. Quantification of gene transcripts under several conditions and by changing several other factors is an important application. Many improved and sensitive disease diagnostic methods can be developed using qrt-PCR.