Introduction

We will mark with fluorescein Taqman probe and template DNA after mixing, complete heat denaturation and renaturation at low temperature, due to extension of thermal cycle, and abide by the law of polymerase chain reaction, and the template DNA complementary pairing Taqman probe is cut off, fluorescein free in the reaction system, fluoresce under certain excitation light, with the increase of cycling times,??The amplified target gene fragment increases exponentially. The Ct value can be obtained by real-time detection of the corresponding fluorescence signal intensity that changes with the amplification. Meanwhile, the copy number of the target gene in the tested sample can be obtained by using several standard samples with known template concentration as the control.??

1 Method

1.1 SYBRGreen

In the PCR reaction system, excessive SYBR fluorescent dye is added, SYBR fluorescent dye is specifically doped into the DNA double chain and emits fluorescence signal, while SYBR dye molecules in the chain will not emit any fluorescence signal, so as to ensure that the increase of fluorescence signal is completely synchronized with the increase of PCR products. Fluorescence curve of SYBR quantitative PCR amplification. Fusion curve of PCR product (single peak diagram shows the singleness of PCR amplification product)

1.2 TaqMan probe

When the probe was intact, the fluorescence signal emitted by the reporting group was absorbed by the quenched group. During PCR amplification, the 5 '-3' exonuclization activity of Taq enzyme degrades the probe enzyme and separates the reporting and quenching fluorophore groups, so that the fluorescence signal can be received by the fluorescence monitoring system, that is, for every AMPLIFIED DNA strand, a fluorescence molecule is formed, realizing the complete synchronization of the accumulation of fluorescence signal and the formation of PCR products.