Characteristics of the PCR technology reaction
What is PCR technology?
Polymerase chain reaction (PCR) is a kind of molecular biological technology used to amplify specific DNA fragments, which can be regarded as the special DNA replication in vitro. The biggest characteristic of PCR is that it can greatly increase the trace amount of DNA. So if a tiny bit of DNA can be isolated, be it from a fossil, the remains of a historical figure, or hair, skin or blood left by a murderer decades ago, PCR can be used to amplify and compare it.
How does PCR technology works?
PCR uses DNA denaturation at 95° Celsius in vitro to become single strand. At low temperature (usually around 60°C), primer and single strand are combined according to the principle of base complementary pairing. Then the temperature is adjusted to the optimal reaction temperature of DNA polymerase (around 72°C), and DNA polymerase synthesizes complementary strand along the direction of phosphoric acid to five-carbon sugar (5'-3'). The polymerase based PCR device is actually a temperature control device, which can be well controlled between denaturation temperature, renaturation temperature, and extension temperature.
Characteristics of the PCR reaction
(1) Strong specificity
The specific determinants of PCR reactions are:
1.Specific and correct binding of primer and template DNA;
2.Base pairing principle;
3.The fidelity of Taq DNA polymerase synthesis reaction;
4.Specificity and conservation of target genes.
The correct combination of primer and template is the key. The binding of primer and template and the extension of primer chain follow the principle of base pairing. The fidelity of polymerase synthesis reaction and the high temperature resistance of TaqDNA polymerase enable the binding (renaturation) of template and primer in the reaction to be carried out at a high temperature, the specificity of binding is greatly increased, and the amplified target gene fragment can maintain a high accuracy. By selecting target gene regions with high specificity and conservation, the degree of specificity will be higher.
(2) High sensitivity
The amount of PCR product produced increased exponentially, allowing the initial template under test to be amplified to microgram (μg=-6) levels on the order of picogram (pg=10-12). One target cell can be detected from a million cells; The sensitivity of PCR for virus detection was up to 3 RFU(plaque forming unit). The minimum detection rate in bacteriology was 3 bacteria.
(3) Simple and fast
The PCR reaction was performed by Taq DNA polymerase with high temperature resistance. After the reaction solution was added once, the denature-anneal-extension reaction was carried out on the DNA amplification solution and water bath, and the amplification reaction was generally completed in 2 to 4 hours. Amplification products are generally analyzed by electrophoresis, not necessarily by isotope, without radioactive contamination and easy to popularize.
(4) Low purity requirements for specimens
There is no need to isolate viruses or bacteria and culture cells. Both crude DNA and RNA can be used as amplification templates. Can directly use clinical specimens such as blood, coelomic fluid, washing fluid, hair, cells, living tissue DNA amplification detection.
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