The so-called real-time quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve.
At present, real-time quantitative PCR technology mainly uses two methods: fluorescent dyes or fluorescently-labeled probes to label and track PCR products, real-time online monitoring of the reaction process, and combined with corresponding software to analyze the product and calculate the sample template to be tested. the initial concentration.
1. Fluorescent dye method
In the PCR reaction system, an excess of fluorescent dye is added. The dye only binds to the minor groove of double-stranded DNA, not to single-stranded DNA, and does not emit fluorescence in the free state. It can emit light only when it is incorporated into the double-stranded DNA. Therefore, in the PCR system, with the exponential amplification of specific PCR products, the dye is incorporated into the double-stranded DNA during the extension phase of each cycle, and its fluorescence signal intensity is positively correlated with the number of PCR products.
Fluorescent dyes include saturated fluorescent dyes and unsaturated fluorescent dyes. The typical representative of unsaturated fluorescent dyes is the most commonly used SYBR Green I. EvaGreen and SolisGreen are saturated fluorescent dyes. Unsaturated fluorescent dyes will inhibit the PCR process when the concentration is high, so a slightly lower concentration is used in fluorescence quantitative experiments, and the dye cannot occupy all positions on the DNA double-strand. When the PCR temperature rises, the double-strand gradually melts, and the dye will fall off the unwound single-strand, and then bind to the adjacent unmelted double-strand to continue to fluoresce. This dye rearrangement results in that, although the melting process of the DNA double-strand is different, the fluorescence value is almost constant within a small temperature change, that is to say, the melting curve cannot accurately reflect the melting of the double-strand.
Therefore, for unsaturated dyes, the resolution of the dissolution curve is relatively low, and only specific products can be distinguished, but the differences of single bases cannot be distinguished. Saturated fluorescent dyes will have dye molecules in all the binding sites in the DNA double-strand, and the binding of the dye to the DNA is in a saturated state. There are no sites in the chain portion where the dyes can bind, and the dyes cannot rearrange. Therefore, the resolution of the melting curve made by saturated dyes is very high, which can accurately reflect the melting of DNA double-strands with temperature, and the accuracy can reach the distinction of single-base differences.
2. Fluorescence-labeled probe method
During PCR amplification, a specific fluorescent probe is added together with a pair of primers. The probe is a linear oligonucleotide with a fluorescent reporter group and a fluorescent quencher group labeled at both ends. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. PCR During PCR amplification (in the extension stage), the 5’→3′ exonuclease activity of Taq enzyme cleaves and degrades the probe, so that the reporter fluorophore and the quencher fluorophore are separated, thereby The fluorescence monitoring system can receive the fluorescence signal, that is, every time a DNA chain is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescence signal is completely synchronized with the formation of the PCR product, which is also the basis of quantification.
3. Dye method VS probe method
1. The probe method can increase the specificity of the signal collected by the reaction through the probe. The signal can only be collected when the fragments bound by the probe are amplified. The probe method can use multiple system reactions, and can predict and optimize the reaction conditions in advance. , the disadvantage is the high cost of synthesizing probes.
2. The dye method is economical and can do the dissolution curve and analyze the TM value of all PCR products. The disadvantage is that the specificity is not as good as that of the probe method.
