: Patients with a particular form of hemophilia (a deficiency in blood clotting)

Question

: Patients with a particular form of hemophilia (a deficiency in blood clotting) have a loss of an EcoRI restriction site within the gene for a coagulation factor protein. In one family with an affected son, PCR analysis was carried out on blood samples from a male fetus and from several family members to determine whether the fetus carried the mutation. Following PCR amplification of the appropriate 150 bp (base-pair) DNA fragment, the fragments were incubated with EcoRI and the resulting cleavage fragments separated by electrophoresis on an agarose gel and stained with ethidium bromide.

Will the fetus have hemophilia? Explain. Given that the EcoRI restriction digest is "complete" (i.e. all DNA that can be cut is cut), explain why there are 3 bands in the Mother's lane. This analysis can also be done by using Southern blotting to detect single-copy sequences in genomic DNA. Why is the PCR analysis preferrable (i.e. what are the benefits of PCR vs. Southern blotting)?

Explanation / Answer

a) The fetus will not have hemophilia. The easiest comparison is to look at the "normal male control", which shows only two bands, at 100bp and 50bp. This indicates that the gene is cleaved into these two segments by the EcoRI. Similarly, the Fetus shows these same two bands (and no band at 150bp). Clearly, the fetus will not have the disease.

b) The easiest comparison is to look at the heterozygous control. In this lane, there are three bands: one for the uncut 150bp segment, and two for the fragments after being cut by EcoRI. Obviously, when the genes are heterozygous (Xx), and contain the recessive gene, then not all of the fragment is cut, and there will be all three of these bands. The mother is obviously heterozygous as well, which explains these bands.

c) The advantage to the PCR in this case is the ability to see not just the 150bp uncut fragment, but also whether there will be the 100bp and 50bp fragments as well. The presence of the bands can indicate the genotype of the individual, as follows:

150bp Fragment Only: Affected Individual

100bp/50bp Fragments Only: Unaffected (Homozygous) Individual

All Three Fragments: Unaffected (Heterozygous) Individual

Southern blotting will not allow you to ascertain this information, only the presence of the single-copy sequence.

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