The principle of PCR is to use DNA that denatures and becomes single-stranded at a high temperature of 95°C in vitro. At low temperatures (usually around 60°C), the primers and single-strands are combined according to the principle of base complementary pairing, and then the temperature is adjusted to DNA polymerase. At the optimum reaction temperature (around 72°C), DNA polymerase synthesizes complementary strands along the direction of phosphoric acid to five carbon sugars (5'-3').
The most valuable application area of PCR is the diagnosis of infectious diseases. In theory, as long as there is a pathogen in the sample, PCR can be detected.
In experiments, it was found that DNA can also be denatured and melted at high temperatures, and it can be renatured into double strands again when the temperature is lowered. Therefore, by controlling the denaturation and renaturation of DNA by temperature changes, adding design primers, DNA polymerase and dNTPs can complete the in vitro replication of specific genes.
However, DNA polymerase will be inactivated at high temperatures. Therefore, new DNA polymerase must be added in each cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology.
