Real-time fluorescence quantitative PCR

Real-time fluorescent quantitative PCR(Quantitative Real-time PCR, referred to as qPCR) is a method used in PCR reaction systems where fluorescent substances are added. It utilizes the accumulation of fluorescent signals to monitor the entire PCR process in real-time and,in the end, quantitatively analyzes the unknown template through the standard curve.
Fluorescent substances used in real-time fluorescence quantitative PCR can be divided into Two types:
1. Fluorescent probe: The probe is an oligonucleotide, and both ends are respectively labeled with a reporter fluorescent group and a quenching fluorescent group. When a specific fluorescent probe is added to the PCR reaction system, when the probe is complete, the fluorescent signal emitted by the reporter group will be absorbed by the quenching group, and the fluorescence detection system cannot receive the fluorescent signal. During PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, separating the reporter fluorescent group from the quenching fluorescent group, thus the fluorescence monitoring system can receive the fluorescent signal, that is, for each amplified DNA strand, a fluorescent molecule is formed, and the accumulation of fluorescent signals is completely synchronized with the formation of PCR products.

 


2. Fluorescent dye: SYBR fluorescent dye is added to the PCR reaction system. SYBR fluorescent dye can be non-specifically incorporated into DNA double strand to emit fluorescent signal, while SYBR dye molecules not incorporated into the chain will not emit any fluorescent signal, so as to ensure that the increase of fluorescent signal is completely synchronized with the increase of PCR products. SYBR binds only to double-stranded DNA, so the specificity of the PCR reaction can be determined by the dissolution profile.

 


Real-time fluorescence quantitative PCR technology can be applied in many fields, including: pathogenic microorganism detection, tumor early screening, tumor detection, gene mutation analysis, NIPT, genetic disease screening, etc.


 

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