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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?


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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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