Primer Design Principles
Taqman primer design principle
1. Determine the probe before designing primers.
2. When designing primers, get as close to the probe as possible without overlapping the probe.
3. Avoid using 4 or more consecutive G.
4. The Tm value of each primer should be 58-60°C.
5. The last 5 nucleotides at the end of the primer cannot have more than 2 G and C.
6. Primers had better not contain self-complementary sequences, otherwise they will form the hairpins.
7. In order to avoid the amplification of the genome, it is best to design primers across exons.
8. The length of amplification product should be 50-150 bp in order to obtain the best PCR efficiency.
9. No other non-specific products were found in the comparison results on NCBI.
Taqman probe design principle
1. Probe length should be 13-25 bp (13-30 bp if conventional TaqMan probe is used).
2. The Tm value should be 65°C~70°C, which is usually 5°C~10°C higher than the TM value of the primer to ensure that the probe preferentially binds to the target gene during
3. For a primer, the content of guanine-cytosine (G+C) should be between 40% and 70%.
4. The 5 'end of the probe should avoid using G, because the 5' end G will have quenching effect, even if it is cut off.
5. In the whole probe, the content of C is obviously higher than that of G, and the high content of G will have quenching effect, so we can choose another paired chain as the probe.
Taqman MGB probe design principle
1. A report dye (for example, FAMTM) is attached to the 5 'end of the probe.
2. There is a non-fluorescence quenching group (NFQ) at the 3 'end of the probe.
3. The part of MGB is attached to NFQ, and MGBs increases the annealing temperature (Tm) without increasing the length of the probe, so a shorter probe can be designed, but not less than
4. In principle, as long as there is a base mutation in the MGB probe, MGB can detect it (the MGB probe will not bind to the target gene and will not produce a fluorescent signal).