Real Time PCR. The idea of the method and strategies of reaction monitoring
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Real-time PCR has become one of the most widely used methods of gene quantitation in molecular biology and medical diagnostics. This technique combines PCR amplification and the detection of the PCR product into a single step. In real-time PCR, the amount of product formed is measured during the course of the experiment by monitoring the fluorescence of dyes or probes introduced into the reaction. Fluorescence data are generated by the use of intercalating dyes such as SYBR Green I or molecular probes, the most important of which are: TaqMan, Molecular Beacons, Hybridization Probes, and Scorpion Probes. The real-time PCR reactions are characterized by the PCR cycle in which the target amplification is first detected. This cycle is referred to as a treshold cycle (Ct), at which fluorescence intensity becomes greater than background fluorescence. Consequently, the greater the quantity of target DNA in the starting material, the faster the significant increase in fluorescence intensity will appear, yielding lower Ct. The relation between Ct and the concentration of the target sequence allows for a precise quantitation of the genetic material in the sample.
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Jaroslaw Tyburski, Zaklad Biotechnologii, Wydzial Biologii i Nauk o Ziemi, Instytut Biologii Ogolnej i Molekularnej, Uniwersytet Mikolaja Kopernika, ul. Gagarina 9, 87-100 Torun, Poland