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qPCR Basics

The Polymerase Chain Reaction (PCR), first described by Mullis and Saiki in 1985, has made it possible to detect rare target nucleic acid sequences isolated from cell, tissue or blood samples. Real-time quantitative PCR (qPCR) is a powerful improvement of the basic PCR technique. The use of fluorescent detection strategies in combination with appropriate instrumentation enables accurate quantification of nucleic acids. Quantification and identification of nucleic acids is achieved by measuring the increase in fluorescence during the exponential phase of PCR.

 

 

 
fig A
fig B

 

The point at which the fluorescence rises significantly above background is called the threshold cycle, Cq (Fig. A). There is an inverse linear relationship between the log of the starting amount of template and the corresponding Cq-value during qPCR (Fig. B).
 

Compared with endpoint quantification methods, real-time amplification assays offer reproducible results and have a much wider dynamic range. In addition, the use of fluorescent agents and probes that only generate a fluorescence signal upon binding to their target enables real-time amplification assays to be carried out in sealed tubes, eliminating the risk of carryover contamination.

In qPCR, two types of fluorescence detection chemistries are utilized:
1)    Intercalating dye-based chemistry: a fluorescent dye (e.g. SYBR Green) intercalates and binds within the double-stranded PCR product
2)    Probe-based chemistry: the fluorescent–labeled target-specific probe hybridizes to the PCR product, resulting in increased specificity and sensitivity.