qPCR

qPCR stands for quantitative polymerase chain reaction, a technique that aims to read out the quantity of DNA in a given sample. The core concept of this technique is the use of amplification cycles to detect very small amounts of DNA (up to single strands!), i.e. many copies of the DNA strand to be detected are made.

The basic principle follows 4 steps: the selection of the DNA strand to be detected, heating of the sample to separate the DNA strands, addition of the primers complementary to the target sequences (i.e. the two separated DNA strands), and finally the addition of the polymerase, the enzyme that extends the primers to form new strands of DNA. This process is then repeated until a sufficient amount of strands is produced.

One can also add a non-specific DNA-binding fluorescent dye: the purpose of this dye is to track the amplification cycles as they are happening, or in scientific terms, real-time. As the amount of DNA stands increases every cycle, so will the fluorescence, which is therefore used to monitor the amplification process.

RT-PCR method