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I am designing primers for qPCR from conserved regions of a few different fish species.This is because gene sequence for my fish species are yet unavailable in NCBI database.

Is it OK (can the primer anneal to the target sequence) if the primers have several mismatch at the interior and towards it 5’ end? At the 3’ end, I can only design primer with maximum 5 conserved bases.

Thank you.

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  • $\begingroup$ qPCR will not work reliably with so poorly matched primers, so either create multiple primer sets with higher specificity or create multiple primersets amplifying subsets of the target... this will create multiple better fitting fragments, that will act as optimised primers in later rounds of the qPCR experiment... keep in mind that the latter option will produce quite a lot of background noise. $\endgroup$ – Jeppe Nielsen May 8 '17 at 2:19
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QPCR is extremely sensitive, so I would not do this. Instead I would get those primers you mention in your question and instead sequence using them instead of doing qpcr with them. Then I would make new primers specific for the region you want to do qpcr on based on the sequence.The advantage of this is that you would now know the sequence and so you would have better primers for qpcr.

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