Detailed explanation of the selection of fluorescent labels in real-time quantitative PCR experiments

Fluorescence quantitative PCR technology combines conventional PCR and fluorescence detection technology, and uses fluorescence signal accumulation to monitor the entire PCR process in real time, so as to achieve quantitative analysis of the initial template

When it comes to real-time PCR technology, we often ask questions such as "is your experiment a dye method or a probe method", "what type of probe do you use", etc.

dye method

The dye method utilizes dyes that can bind to DNA double strands, such as SYBR Green

▲ Figure 1 Schematic diagram of dye method

In theory, all dyes that can bind to double-stranded DNA can be used for qPCR detection, such as ethidium bromide EtBr, propidium iodide PI, acridine orange, Cy3,

▶ Little inhibition of PCR reaction

▶ High binding density to DNA

▶ The chemical properties are more stable and suitable for long-term storage

TaqMan Probes

TaqMan probes can basically meet more than 60% of qPCR experiments, such as conventional gene expression and copy number variation CNV experiments

▲ Figure 2 Taqman probe

MGB probe

For SNP experiments to resolve single-base differences, MGB probes with lower tolerance for base mismatches were used, and a DPI3 group was added after the quenching group (Figure 3), thereby improving the binding affinity to the target.

▲ Figure 3 MGB probe

two-hybrid probe

Taqman probes have stricter requirements on the length of probes, and double-hybrid probes eliminate this "defect"

▲ Figure 4 Dual hybrid probe

Molecular beacon probe

In the free state, the molecular beacon probe is a stem-loop structure, the 15-30 bases of the loop part can be combined with the target region, and the paired region at the lower end (generally 5-6 bases on the left and right) It consists of repeated GCs, so that the 5' fluorescent molecule and the 3' quenching group are tightly grouped together, and the fluorescence is quenched; when annealed, the molecular beacon probe unwinds the loop and hybridizes with the template target, so that The physical distance between the fluorescent molecule and the quenching group becomes larger, and the premise of fluorescence quenching is broken (Fig.

▲ Figure 5 Molecular beacon probe

In addition to the above-mentioned types of probes, there are also technologies that try to combine the functions of primers and probes, such as Amplifluor, LUX, etc.
, which are more difficult to design, but easier to use
.
Each has its own application focus and design advantages and disadvantages, so I won't go into details here
.
So in the fluorescence quantitative PCR experiment, how should we choose? Here, we summarize some of the differences between the two methods for your reference
.

Table 1 Differences between dye method and probe method