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What you need to know about qPCR and Real-Time PCR
About qPCR and Real-Time PCR:
Many people confuse RT-PCR with real-time PCR, but they are not the same. We'll talk more about RT-PCR later. Real-time PCR is also known as quantitative real-time PCR, also known as qPCR, which is why they should be the same thing. With the help of fluorescent chemicals, real-time PCR allows us to detect the amplification of DNA in the process of PCR, so that there is no need to run electrophoresis, which greatly simplifies the experimental steps of the operator.
Experimental principle of qPCR:
To put it simply, qPCR is the introduction of fluorophores in the whole amplification process. With the multiplication of DNA, the fluorescence signal in the reaction system will also be enhanced. By detecting the intensity of the fluorescence signal, the DNA amplification was quantitatively analyzed.
At present, we can roughly divide qPCR into two kinds according to different fluorescent substances. One is to use fluorescent dye, mainly SYBRGreenⅠdye, which can bind to DNA double strand, and will display fluorescence signal after binding, but when there is no binding, the fluorescence signal is almost impossible to detect, with the increase of copy number, the fluorescence signal is enhanced. The other is the use of fluorescent probe, also known as TaqMan probe method, whose principle is to design a fluorescent probe that can specifically bind to the target gene according to the target gene. This probe contains two groups: A fluorescent groups and a quenching, normally because of the existence of quenching groups cannot glow fluorescent groups, and the amplification of probe binds to DNA template, and expanded to the probe position two groups are cut, and fluorescent groups can fluoresce, also with the increase of copy number, enhanced fluorescence signal.
With the aid of the quantitative relationship, people can map the PCR amplification curve, the curve is fluorescent signal as the cycle number changing curve, with the aid of the curve can analyze the PCR status, the curve of the ideal should be to present the growth of the "S" type, but about the content of the curve analysis is more complex, here is no need to do too much for the time being. At least we know that with this technique we can analyze our PCR results more accurately without having to go through the electrophoresis after each PCR. Therefore, this technology is more and more widely used.
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