I need to design Taqman probes for different species of the same genus in order to differentiate between them.
I have read the guidelines (no 5' G end on probe, max 2 GC 3' end primers, ~10C difference between probes and primers etc).
My issue is what's more important -t to have highly different primers for each species so that they cannot anneal on the other species DNA or highly different probes?
To rephrase: let's say I found a probe with 2 bp difference (length ~22bp) and primers 3' end difference ~2 bp (primer length 17-20 bp). Would that be acceptable or I need to find a probe with 4 or 5 different bases?
Also would 2 bp on the 3' end of primers difference be acceptable or would it be better to have at least 3 bp ?
in such a case, I would have first searched for primers that anneal to different regions of the template that have sufficiently different sequences between the species to be identified. It is not necessary that the primers are designed to anneal at one particular genetic position. This approach has some benefits- if the amplicon size is sufficiently distinct (~50 bp or more) between the two species, a simple PCR can tell you the species. Even a SYBR green based qPCR can indicate the species based on the difference in the melting curve.
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