RFLP / MstII test for Sickle-Cell Anemia
Sickle-cell Anemia is a
molecular disease caused by a mutation in the beta-globin
gene. The difference between the standard BA allele and
the sickle-cell BS
allele is a single-nucleotide substitution (AT) in the second
position of the sixth codon of this gene. The sequence of the
standard BA allele (CCTGAGG)
happens to correspond to an MstII
restriction site (CCTNAGG), which is
altered in the BS allele (CCTGGG).
gene region includes two flanking MstII sites (red lines).
In the genetic test for the BS allele, total DNA from the individual tested is digested with MstII and run in a Southern Blot. The blot is hybridized with a probe specific for the beta-globin gene. If the standard BA allele is present, the probe sticks to the two small MstII fragments and produces two smaller bands. If the sickle-cell BS allele is present, the probe sticks to the single large fragment and produces one larger band. Thus, a standard AA homozygote shows the two-band pattern, a SS homozygote (with sickle-cell anemia) shows the one-band pattern, and an AS heterozygote (with sickle-cell trait) shows all three bands. The pattern of molecular expression is therefore described as co-dominant.
this particular test depends on the coincidence that the
nucleotide substitution responsible for the sickle-cell allele
happens to occur in such a way as to create an RFLP: the absence of the MstII site does not itself
but is instead a genetic
marker for the allele.
Suppose this experiment were done by amplifying the
beta-globin gene by PCR, then cutting the product with
and separating them by electrophoresis as above. How many bands would be
expected in the heterozygote? Explain. Draw
the expected result.