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A brief discussion on real-time fluorescence quantitative PCR technology

News source: Release time:[2023-09-06]

At present, according to the different fluorescent chemicals used in real-time fluorescent quantitative PCR, fluorescent quantitative PCR technology is mainly divided into two categories, namely fluorescent dyes and fluorescent probes. Fluorescent dyes are a non-specific detection method for amplified sequences and are the earliest methods used in fluorescence quantitative PCR. Fluorescent probes are fluorescent quantitative PCR technology based on the principle of fluorescence resonance energy transfer (FRET).

Fluorescent dye

The fluorescent dye method is also known as DNA binding staining. DNA-binding dyes are the earliest chemicals used in fluorescence quantitative PCR. The main dye molecule currently used is SYBR Green I. SYBR Green I can specifically bind to the minor groove of DNA double strands. Free SYBR Green I has almost no fluorescence signal, but after binding to DNA, its fluorescence signal can increase hundreds of times. Therefore, the more products amplified by PCR, the more SYBR Green Ⅰ is bound, and the stronger the fluorescence signal will be [1], which can quantify any target gene.

The basic principle of SYBR GreenⅠluminosity

Hydrolysis probe

Hydrolysis probes are represented by TaqMan probes, also known as exonuclease probes. TaqMan technology is a kind of real-time fluorescent quantitative PCR technology researched and developed by American PE company in 1996, and it has been widely used in the quantitative detection of genes [2,3]. The basic principle is to use the 5' exonuclease activity of Taq enzyme, that is, Taq enzyme has natural 5'-3' exonuclease activity, which can cleave nucleotides at the 5' end of double-stranded DNA and release a single oligonucleotide acid.

Comparison of advantages and disadvantages of methods

Fluorescent Dye

1. It is easy to use and does not require complex fluorescence design, making the detection method simple and reducing the cost of 


2. It can monitor the amplification of any double-stranded DNA sequence, has no primer specificity, 

and can be used for different templates

Since fluorescent dyes can bind to any double-stranded DNA, they 

can also bind to non-specific double-stranded DNA (such as primer-dimers), making the experiment prone to false positive signals.

The primer-dimer problem can currently be solved using melting curves to distinguish between specific and non-specific amplification.
Hydrolysis Probe

1. It solves the disadvantage of non-specificity of fluorescent dyes, and does not need to analyze the melting curve of oligonucleotides after the reaction, which shortens the experimental time.

2. Because TaqMan probes have 

high specificity to the target sequence, they are especially suitable 

for SNP detection.

1. The price of TaqMan probe is relatively high, and it is only suitable 

for a specific target, which is inconvenient for popularization and application.

2. Since the fluorescent groups and quenching groups on both sides of the TaqMan probe are far apart, the quenching is incomplete and the background is high. Moreover, this method is also susceptible to the 5'-3' exonuclease of Taq DNA polymerase. activity impact.

The new MGB-TaqMan probe reduces the interference of background signals.

Technical principles of SpaceGen products:

After a series of transformations, unmethylated cytosine (C) can be converted into uracil (U). After PCR reaction, uracil (U) is finally converted into thymine (T), that is, unmethylated cytosine (C). Pyrimidine (C) will eventually be converted into thymine (T); methylated cytosine (C) will not change during the modification reaction due to the protective effect of its methyl group, that is, methylated cytosine (C) ) is converted to cytosine (C).

SpaceGen’s methylation detection products use PAP-ARMS® technology to complete the fluorescent quantitative PCR detection of the modified genome, and combine the pyrophosphate hydrolysis activation reaction on the traditional ARMS technology principle. The TaqMan probe method and the experimental process of nucleic acid extraction-methylation modification-fluorescence PCR were used for detection.


1、Proc Nat Acad Sci USA,1996,93:14509-14514.

3、Clin Chem Lab Med,1998,36:587-588.

4、Nat Genet,1993,4(3):289-294.