Use of an RFLP as a
genetic marker for a
At a particular gene
locus, alleles A & B share two
lines), but Allele B has
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,
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
the autoradiogram if Allele A is present. If Allele B
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 autoradigoram shows
the presence of the
linked mutation, but does not cause the trait. It
described as a "marker"
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 counselling to
determine if they may be carriers. The genetic
shows that two (II-3 & II-4) are carriers, and that
of the two children of the latter (III-2)
also a carrier.
same experiment were done by amplifying the 5kb region by
PCR, and the products are cut
with the restriction enzyme and separted
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.