Analysis of Experimental Data
Adapted from http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=iga.section.2323

DNA studies are performed on a large family that shows a certain autosomal dominant disease of late onset (approximately 40 years of age). A DNA sample from each family member is digested with the restriction enzyme TaqI and subjected to gel electrophoresis. A Southern blot is then performed, with the use of a long radioactive probe consisting of part of the studied gene .

How many copies of the studied gene do you expect to find in each sample?

The family pedigree and the Southern blot autoradiogram are shown below. Affected members are shown in black.


 

What is the DNA restriction profile of the healthy family members (shown in white)?
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

What is the DNA profile of most affected family members?
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

How can using a different probe affect the results?



 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
How can you explain the case of the last son who is affected by the disease but has only the 5Kb DNA fragment?













































This is an autosomal disease, so both males and females are expected to have two copies of the studied gene. However,  the healthy family members have the dd allele combination, whereas the affected family members have the Dd allele combination.

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All healthy family members (dd) have only the 5Kb band. The probe hybridizes to a sequence ~ 5Kb between the two TaqI  recognition sites. (The probe can also slightly protrude beyond one of the TaqI cleavage sites.)

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Allaffected family members have the Dd allele combination. The D allele  bears an additional TaqI site in most cases, resulting in the formation of two smaller fragments of 2Kb and 3Kb, instead of the 5Kb fragment characteristic of the d allele.
Since the probe used is long enough, it can still hybridize with the two shorter TaqI fragments of the D allele under appropriate conditions.
 


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Using a different probe (e.g. a shorter one) may affect the pattern of recognised DNA fragments on the Southern blot. Perhaps only one fragment will be recognized although the same two fragments are still being formed. (Recognition will depend on the probe and on the hybridization conditions, such as ionic strength of the hybridization buffer etc.)
Thus, we see that the "lack" of one fragment in the southern profile does not necessarily reflect a deletion, it can reflect a point mutation just as well.
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The last son most likely represents a case of crossover between the WT d allele and the disease locus on the D allele. This  crossover results in the loss of the extra TaqI site on the D allele, but the defective form of the gene is still present. Thus, we observe a  5Kb DNA fragment derived from an affected family member.
Since the 3- and 2-kb fragments are linked to the disease allele, the presence of these fragments can be used as a predictor for the disease. However, the diagnosis has to take into account the possibility of a crossover.
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