How can you identify if a person is homozygous for a certain allele?

Question

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?

Answer 1

Do you know why primers would not be able to differentiate between the mutant and wild type ("normal") gene?

If the reason is because the wild type and normal gene are a very similar size (and therefore would not separate on a gel), then perhaps there is a recognition site for a restriction enzyme in one version of the allele that is not present in the other version. For example, if the wild type allele does have a restriction site and the mutant allele does not, then you can digest a person's DNA sample with the proper restriction enzyme after amplifying the gene. After digestion, you can "clean" your DNA using a kit and run your samples through a gel. People that are homozygous for the wildtype allele should show two bands (since their allele got cut with the restriction enzyme), people that are homozygous for the mutant allele should show one big band (uncut allele), and people that are heterozygous should show one big band (the mutant uncut allele) and two smaller bands (the cut wild type allele).

To find restriction sites in your sequences, you can download the free ApE software, copy and paste the sequence of your mutant allele in one window and the sequence of your wild type allele in another window, and for each sequence you can click "Enzymes" and then "Enzyme Selector" and it will show you a list of enzymes with the number of restriction sites found in your sequence. To see exactly where the restriction site is, you can click on the enzyme and click "Highlight." Then you can see how long your bands should be if the allele is digested.