1.Choose the probe first, and then design the primers so that they are as close to the probe as possible.
2.The chosen sequence should be highly specific, and the amplified fragment with the smallest secondary structure should be selected as much as possible—this can affect the efficiency of the reaction and hinder the amplification of the enzyme. It is recommended to test the secondary structure first, and if the secondary structure cannot be avoided, then the annealing temperature should be increased accordingly.
3.The amplification length should not exceed 400 bp, the ideal amplification length is within 100-150 bp, and the shorter the amplification fragment, the easier it is to obtain an effective amplification reaction. Shorter amplified fragments also make it easy to ensure consistency in the analysis.
4.Keeping the GC content between 20 and 80 is susceptible to non-specific reactions in GC-rich regions, which can lead to reduced amplification efficiency and non-specific signals in fluorochrome analysis.
5.To ensure efficiency and reproducibility, duplicate nucleotide sequences, especially G (there cannot be 4 consecutive Gs).
6.Primers and probes are paired with each other to avoid the formation of dimers and hairpins.
In principle, it is relatively similar to primer design. Just because the probe sequence requires a high degree of matching, there are strict requirements for the GC content and TM value of the probe, and it is also necessary to consider matching with upstream and downstream primers. If you already have a dye primer at hand, and then you want to still use this pair of primers, can you choose a piece in the middle of the sequence corresponding to this pair of primers as a probe?We don't recommend it. Because the upstream and downstream primers you design separately are fine, and the probes that are individually designed look fine, but when you put the primers and probes together, there is a risk that the parameters are incompatible. So it's better to put the three together and design it.
ELISA assay kits