Molecular Pathology- Miscellaneous I
Question No. 30. The Answer is: (A) Wildtype A / heterozygous B with a novel polymorphism that creates an additional restriction site.
Discussion: In gel electrophoresis, smaller molecules move faster and large molecules move slower. Within the same period of time of electrophoresis, the smaller molecules will be farther away from the origin (well) than the larger molecules.
In this particular question, Lane 2 reveals a larger fragment and a smaller fragment after restriction enzyme digestion. So, there must be one restriction site in the PCR product which is invariant (i.e., not polymorphic).
Lane 3 tells you that the polymorphism A leads to acquisition of another restriction site that cuts the smaller of the two fragments. There are two possibilities, either the smaller fragment is cut into two smaller fragments (a and b) that are of similar molecular weights or into two fragment of dissimilar size and the smallest of these has run off of the end of the gel.
Lane 4 tells you that the polymorphism B leads to acquisition of another restriction site that would allow cutting the larger fragment into two smaller fragments (g and d), again either of similar molecular weights or the smaller fragment has run off of the end of the gel.
Lane 5 tells you that the polymorphism leads to the acquisition of a new restriction site that would generate a larger fragment (l) and a very small fragment (t). Since l is larger than A, it has to come from the B fragment. Since t is smaller then (a and b), the location of this new restriction site has to be on fragment B and very close to fragment A.
The rationale is summarized in the following schematic diagram.