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I've been thinking about starting a small private research project. In this project I need to find out whether a person is homozygous for a certain allele. The reason for this is that I'm really trying to find out whether has a mutation that makes him/her not produce this chemokine. Thus I need to identify in an easy way which people are "normal", which people have the mutation in one chromosome, and which people have the mutation in both. About 1% of the population have the mutation in both chromosomes.

A primer would AFAIK not be able to differ between the heterozygous and the "normal" people.

Do anyone know how to approach this?

Pedery
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1 Answers1

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You could use a 3-primers strategy, where you have a common primer, one that only amplifies the mutant allele and one that only amplifies the wt. This may or may not be feasable depending on the type of mutation. DNA from het people will amplify both product, while homozygous will only amplify one (wt or mutant).

Of course then, linking the mutation of a gene to the lack of a chemokine is not necessarily straightforward.

To be honest, however, I would use a different approach: take people who don't produce the chemokine, and people who do (simply by dosing the chemokine in the blood). Now sequence the gene in the two groups and look for mutations.

nico
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  • Yeah, that's a smart approach. But I don't think (= don't know if) the nonfunctional gene will look the same in all individuals. Since it's a missense/nonsense mutation, it probably doesn't have a clear characteristic. Anyway, good point! +1 – Pedery Dec 15 '11 at 21:39
  • @Pedery: well, then wouldn't the sequencing be the best strategy in that case? Do you have any information whatsoever on the mutation or its product? – nico Dec 15 '11 at 21:42
  • Not at the moment. Right now I'm trying to get an overview. Sequencing would not really be an option cause I want to make this into a super easy testing-kit, one that would need no expensive machinery and would get a test result from a mere droplet of blood. But indeed, your answer was helpful. If I don't get any better answers, I'll mark yours as accepted. – Pedery Dec 15 '11 at 23:36
  • Btw, what would be the purpose of the common primer? If you have the two other primers, they will still tell you all the information you need, right? – Pedery Dec 17 '11 at 15:50
  • Nope, you need two primers for doing a PCR amplification. If you only have a primer that goes on the mutant allele and one that goes on the wt you will have no way of amplifying anything, as the two will never bind on the same DNA. – nico Dec 18 '11 at 09:49
  • Ah, got it. I was thinking in sets of two primers per allele as one primer. – Pedery Dec 18 '11 at 13:47