RFLP / MstII test for Sickle-Cell Anemia
Sickle-cell Anemia is a molecular
disease caused by a mutation in
beta-globin gene. The
difference between the standard BA
allele and the sickle-cell BS allele
substitution (AT) in
second position of the sixth codon of this gene. The sequence of the
standard BA allele (CCTGAGG)
correspond to an MstII
site (CCTNAGG), which is altered in the BS
beta-globin gene region includes two flanking MstII sites
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.
test depends on the coincidence
that the nucleotide
responsible for the sickle-cell allele happens to occur in such a way
as to create
an RFLP: the absence of the MstII site
but is instead a genetic
marker for the allele.
were done by amplifying
beta-globin gene by PCR, then cutting the product with MstII and
separating them by electrophoresis as above. How many bands would be expected in
the heterozygote? Explain.
Draw the expected result.