Fluorescence quantitative PCR technology

Next, let's learn about the main methods of fluorescence quantification and how to choose the appropriate method. Fluorescent chemistry used in fluorescent quantitative PCR can be divided into two types: fluorescent probes and fluorescent dyes. Fluorescence quantitative PCR methods can be divided into two categories: specificity and nonspecific. The specific detection method is to detect the products with gene-specific oligonucleotide probes labeled with fluorescent dyes in PCR reaction. However, the non-specific detection method is to add excessive fluorescent dyes to the PCR reaction system, and the fluorescent dyes will emit fluorescent signals after being specifically mixed with DNA double strands. The former has higher specificity due to the addition of probe identification steps, but the latter is simple and easy to implement.

Nonspecific SYBR Green I staining method

SYBR Green I is a dye with green excitation wavelength (binding diagram), which binds to all dsDNA double helix groove regions. In the free state, SYBR Green I emits weak fluorescence, but once it binds to double-stranded DNA, the fluorescence is greatly enhanced (action mechanism diagram). Therefore, the fluorescence signal intensity of SYBR Green I is related to the number of double-stranded DNA, and the number of double-stranded DNA in PCR system can be detected according to the fluorescence signal.

Specific Taqman hydrolysis probe method

Taqman fluorescence quantitative technology is based on Taqman fluorescence probe, which is an oligonucleotide with fluorescence emitting group and fluorescence quenching group at both ends. When the probe is intact, the fluorescence signal emitted by the emitting group is absorbed by the quenching group;

In the process of PCR amplification, the exonuclease activity of 5'-3' nucleic acid of Taq enzyme degrades the probe, separating the fluorescence emitting group from the fluorescence quenching group, so that the fluorescence monitoring system can receive the fluorescence signal, that is, each DNA strand is amplified to form a fluorescent molecule, and the accumulation of the fluorescence signal is completely synchronized with the formation of PCR products. So as to realize quantization (as shown below). Commonly used fluorescent groups are fam, Tet, vic and hex.