I am designing primers for real-time pcr for both dyes and probes based chemistry. But I am having problems designing the probe. The probes have secondary structures that went beyond the recommended values suggested below:
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
I've plugged in the Tm of the probe to be 8-10 degrees higher than Tm of primer, and length 15-30 bases. But when I checked published probes and primer pairs, the Tm of probe is usually lower or almost equivalent to the Tm of primers and usually shorter than the primers(Tm 52-65, length 18-30).
I have no idea what I should do
Never thrust published primer/probes 100%.
Tm of probe must be 8-10 degrees higher for optimal results. In some cases this not possible and you have to make a compromise. Try to find another location, try to shift the probe a few bases left or right, use LNA (locked nucleic acid) bases in your design. If 60 degree primer and 70 degree probe is not good try 58 and 66 for example. You can even try with a 6 degree difference. (For this you should have at least one reference gene that works reliably under the same condition)
Length of the primer or probe depends on GC content, a GC rich probe may be shorter than a regular primer but in most cases probes are longer. Another possibility is the use of LNAs in the probe which makes it shorter.
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