实时荧光定量PCR原理简述

2020/08/27 admin 31

RT-PCR,全称:Real Time Polymerase Chain Reaction, 实时定量PCR

RT-PCR是PCR技术的创新进步。顾名思义,RT-PCR采用的技术能够在扩增PCR产物时对其进行监控。探测技术是RT-PCR的关键特征。几种探测技术已经商业化:TaqMan探针Molecular Beacon探针。在此,为简洁起见,将描述TaqMan技术(图4.6A )。它代表了一种特殊设计的寡核苷酸探针,其一端附有荧光团,而另一端附有淬灭剂。在退火步骤中将其与模板结合时,由于荧光被猝灭,因此不会发出荧光。但是,在聚合过程中步骤,通过与Taq聚合酶相关的5'至3'核酸外切酶活性切割荧光团,然后可以检测荧光。随着扩增的进行,发出的荧光相应增加。实时监控功能已将PCR从定性转变为定量。例如,平行使用已知拷贝数的标准DNA可以在小于两倍的误差范围内进行定量(图4.6B)。

实时荧光定量PCP原理简述

图4.6。RT-PCR的原理。(A)TaqMan探针。说明了TaqMan探针的原理。注意,从荧光团发出的荧光在被Taq聚合酶除去之前被淬灭。(B)表示通过拷贝数测定的DNA量与扩增周期之间的关系的图。


RT-PCR is an innovative advance of PCR technology. As the name implies, RT-PCR is built with a technology that is capable of monitoring PCR product as it is being amplified. Probing technology is the critical feature of RT-PCR. A few kinds of probing technology have been commercialized: TaqMan probe and Molecular Beaconprobe. Here, for brevity, the TaqMan technology will be described (Fig. 4.6A). It represents a specially designed oligonucleotide probe that has a fluorophore attached to one end, while a quencher is attached at the other end. When it is bound to the template during the annealing step, no fluorescence is emitted, since fluorescence is quenched. However, during the polymerization step, the fluorophore is cleaved by 5′ to 3′ exonuclease activity associated with Taq polymerase, then the fluorescence can be detected. As the amplification proceeds, fluorescence emitted correspondingly increases. The real-time monitoring capability has transformed the PCR from qualitative to quantitative. For instance, the use of a standard DNA with known copy number in parallel allowed quantitation within a less than twofold error range (Fig. 4.6B).


来源:Wang-Shick Ryu, in Molecular Virology of Human Pathogenic Viruses, 2017


实时荧光定量PCR唯一缺点是,它需要配备荧光检测器的特定设备。