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 (CCTGTGG). The beta-globin 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.

    Important: 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 cause sickle-cell anemia, but is instead a genetic marker for the allele.

    Homework: Suppose this experiment were done by amplifying the 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.

All text material 2013 by Steven M. Carr