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 $$ββ^A allele and the sickle-cell $$ββ^S allele is a single-nucleotide polymorphism (SNP) (AT) in the second position of the sixth codon of this gene. The sequence of the standard $β$^A allele (CCTGAGG) happens to correspond to an MstII restriction site (CCTNAGG), which is altered in the $$β$$$β$^S allele (CCTGTGG). The beta-globin gene region includes two flanking MstII sites (red lines).

    In the genetic test for the $β$^S allele, the beta-globin gene region is amplified by PCR. The PCR product is digested with MstII. If the standard $$ββ^A allele is present, the larger fragment will be detected. If the sickle-cell $β$^S allele is present, the the two smaller fragments will be present. Thus, an AA homozygote show a single large fragment, an SS homozygote (with sickle-cell anemia) show two small fragments, and an AS heterozygote (with sickle-cell trait) will show all three fragments. The pattern of molecular expression is co-dominant: both genotype alleles show in the gel phenotype.

    This test depends on the circumstance that the SNP responsible for the sickle-cell allele coincidentally create an RFLP with respect to the standard allele. The SNP in the MstII site does not  itself cause sickle-cell anemia, but is simply a genetic marker for the sickle-cell allele.