Use of an RFLP as a co-dominant genetic marker for a recessive trait

    At a particular gene locus, alleles A & B share two flanking restriction sites (blue lines), but Allele B has an extra restriction site in the middle of the gene. The middle restriction site in Allele B is genetically linked to a DNA mutation (red line) that produces a phenotypically recessive trait. [The middle site is linked in cis to the mutation]

    In the RFLP test, total DNA is extracted from the test individual, and run out in a Southern Blot. A radioactive probe is hybridized to the blot. The probe will anneal to the 5' end of the region: this will identify a 5Kb fragment in the autoradiogram if Allele A is present. If Allele B is present, the middle restriction site cuts off 3Kb from the right-hand 3' end of the fragment, so that the probe lights up only the 2Kb fragment. The 3Kb fragment, which includes the mutation, will not be seen. It is important to realize that the 2kb fragment in the autoradigram shows the presence of the linked mutation, but does not cause the trait. It is therefore described as a "marker" for the trait.

    In the pedigree, birth of individual II-2 with the recessive phenotype indicates that the parents must both be heterozygotes: the other unaffected sibs seek genetic counseling to determine if they may be carriers. The genetic tests shows that two (II-3 & II-4) are carriers, and that one of the two children of the latter (III-2) is also a carrier.

    Homework: Suppose this same experiment were done by amplifying the 5kb region by PCR, and the products are cut with the restriction enzyme and separated by electrophoresis, and that the mutation occurs 2.5 kb from the middle restriction site and 0.5 kb from the right-hand restriction site. How many bands would you expect to see in the heterozygote? Draw the expected gel result. Is this result the same or different than the previous homework assignment? Explain.

All text material ©2016 by Steven M. Carr