Hi Yiwei,

Do you mean expressed genes?

Custom design of Taqman primers is always annoyingly finicky. I can go into more detail how we do this, except I'm a bit confused by what you say you're doing. To design Taqman primers that would work in multiple species would be exceedingly difficult and I'm not aware of too many cases this would work unless you have the level of conservation one may be able to find between say human and chimpanzee. What gene do you think might have a high enough conservation to be able to design cross-species specific primers/probes? Realtime PCR is very sensitive to primer efficiency and is usually quite intolerant of mismatches, which is the beauty of it when specificity is desired.

Hi,

First of all, you should find some genes you can target: present in all species you want to detect (using Uniprot for example). Then you should find the nucleotide sequences of all species (using Genebank) and align them (you can use MAFFT for example). You can also see if that gene is present in some other species you don't want to detect and align them as well. Then identify some conserved region to design your primers and probe (these regions need to be not conserved in the other species). You'll probably need to use some degenerate nucleotide to be more sensitive. Then, you can "Blast" the primers to make sure they are specifics for that gene in all wanted species only.

Finally, you can modify them a little (around the 5' extremity) to have perfect match Tm conditions: (primers: 55°C-60°C with max 2°C difference; probe around 68-70°C)

I hope it will be helpful...

Regards, Léonie