Real-time fluorescent quantitative PCR

Quantitative Real-time PCR (qPCR) is a method in which a fluorescent substance is added to the PCR reaction system, allowing real-time monitoring of the entire PCR process through the accumulation of fluorescent signals. Finally, unknown templates are quantitatively analyzed using a standard curve.
The fluorescent substances used in real-time fluorescence quantitative PCR reactions can be categorized into two types: fluorescent probes and fluorescent dyes.
1. Fluorescent Probe: This probe is an oligonucleotide with a reporter fluorescent group and a quencher fluorescent group labeled at its two ends. When a specific fluorescent probe is added to the PCR reaction system, as long as the probe remains intact, the fluorescence signal emitted by the reporter group will be absorbed by the quencher group, and the fluorescence detection system will fail to detect any fluorescence signal. During PCR amplification, the 5'–3' exonuclease activity of Taq DNA polymerase cleaves and degrades the probe, thereby separating the reporter fluorescent group from the quencher fluorescent group. Consequently, the fluorescence monitoring system can detect the fluorescence signal—meaning that for every DNA strand amplified, one fluorescent molecule is generated, thus achieving complete synchronization between the accumulation of fluorescence signals and the formation of PCR products.

 


2. Fluorescent Dye: SYBR fluorescent dye is added to the PCR reaction system. SYBR dye can non-specifically intercalate into double-stranded DNA, thereby emitting a fluorescent signal. On the other hand, SYBR dye molecules that fail to intercalate into the DNA strands will not emit any fluorescence at all. This ensures that the increase in fluorescent signal is perfectly synchronized with the increase in PCR product. Since SYBR only binds to double-stranded DNA, the specificity of the PCR reaction can be confirmed by analyzing the melting curve.

 


Real-time fluorescent quantitative PCR technology can be applied in multiple fields, including pathogen detection, early tumor screening, tumor detection, gene mutation analysis, non-invasive prenatal testing (NIPT), and genetic disease screening.


 

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