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I'm not quite clear what the question is asking, as far as I can see, one pair would be enough.. Since your primers don't include the distinguishing base (and it would be dicey to e expect a PCR reaction to totally fail because of a one base difference) I assume you are going to do sequencing or digest after the PCR to distinguish between the species?


You do need two pairs of primers one unique to each species as you need a positive result to be confident that the thing you are looking for (cow or horse) is there. There is no limitation on them being in the same place (with a different last base). Usually this would be analyised by gel-electrophoresis and not by sequencing so you don't have to worry ...

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