Question

Exon 4 of the HFE gene from a patient suspected to have hereditary hemachromatosis was amplified by PCR. The G to A mutation, frequently found in hemachromatosis, creates an RsaI site in exon 4. When the PCR products are digested with RsaI, what results (how many bands) would you expect to see if the patient has the mutation and no other RsaI sites are naturally present in the PCR product?

Answer 1

Using PCR and RFLP to detect the hemachromatosis mutation, a homozygous mutation would result in two bands after gel electrophoresis, while a heterozygous mutation would result in three bands.

Step-by-step explanation:

When analyzing exon 4 of the HFE gene for a hemachromatosis mutation using PCR and RFLP, if the patient has the G to A mutation that creates an RsaI site, and assuming no other RsaI sites are present within the PCR product, one would expect to see different band patterns based on the zygosity of the individual. If the individual is homozygous for the mutation, only two DNA bands would be evident after gel electrophoresis one corresponding to the undigested PCR product and one from the digestion at the mutation site. In contrast, a heterozygous individual would show three bands—one undigested and two from digestion at the normal and mutant allele RsaI sites respectively.