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 autoradiogram
shows the presence of the linked
mutation, but does not cause the
trait. It is therefore described as a "marker"
for the trait.
An alternative technical approach is to use the
Polymerase Chain Reaction to amplify a region that includes
the 5kb region between the two outside restriction sites. The
product is cut with the same restriction enzyme as before, and run
out in an electrophoretic gel. In the absence of the mutation, a
5kb band is expected. If the mutation is present, a pair of 2kb
and 3kb bands will be seen. This procedure is much faster, and
avoids the use of radioactive DNA probes.
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 the PCR test
outlined above is used, 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. Explain.