Real Time PCR. The idea of the method and strategies of reaction monitoring

Studzińska, Anna; Tyburski, Jarosław; Daca, Patrycja; Tretyn, Andrzej

doi:0860-7796

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PCR w czasie rzeczywistym. Istota metody i strategie monitorowania przebiegu reakcji Studzińska, AnnaTyburski, JarosławDaca, PatrycjaTretyn, Andrzej

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PCR w czasie rzeczywistym. Istota metody i strategie monitorowania przebiegu reakcji

Real Time PCR. The idea of the method and strategies of reaction monitoring

Studzińska, Anna ; Tyburski, Jarosław ; Daca, Patrycja ; Tretyn, Andrzej

Komitet Biotechnologii PAN ; Instytut Chemii Bioorganicznej PAN

2008

biotechnologia

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 1 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 Cf The relation between Ct and the concentration of the target sequence allows for a precise quantitation of the genetic material in the sample.

Biotechnologia, vol.80, 1 (2008)-.