Choice of primers for PCR

Question

This exercise was given by my professor but I am struggling to understand the solution.

A PCR is performed on the following sequence (in order to replicate the chain and thus have a greater quantity of DNA so to be able to analyze it):

The main sequence belongs to a horse and differences are indicated below with dna belonging to a cow.

1) Choose appropriate primers in order to analyze some lasagna, which is suspected to contain both horse and beef. Given Solution: The primers should be:

I understand how these primers would work in this case, but why not just have the second pair of primers which would make it possible for the whole chain to be analyzed at once?

Answer 1

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 about getting the whole thing. This is because you are only looking for the presence or absence of PCR product.

When you do the PCR you will have 4 reactions, one which amplifies horse only, one which amplifies cow only and their negative controls (adding water instead of lasagna DNA extract). After the PCR you take a small amount of your PCR product and run it on a gel (gel-electrophoresis). If you see amplification in the negative controls then you have contamination and can't trust the result. Otherwise if you see amplification by the cow primers then cow is present in the lasagna and if you see amplification by the horse primers then you probably don't want to eat the lasagna. If you used both options for the first forward primer you suggested (blue) then all your bands would be the same size, if you used both the first (blue) and second (yellow) then you would get different band positions on the gel due to the different sized products which makes you feel more sure that you didn't make the mistake of adding the same primer pair to both tubes (it happens).

In the end it's simply a matter of style.

Answer 2

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?