Southern Blotting in the Diagnosis of Human Disease

                       Southern blotting has been applied to the diagnosis of many human diseases  at the molecular level. These genetic diseases are caused by point mutations, gene rearrangements, or the amplification of genes or specific sequences within the genome. 

                         These methods have in common that restriction-digested genomic DNA is size-separated in agarose gel electrophoresis, transferred onto the membrane, and hybridized with gene-specific probes. Restriction fragment length polymorphism (RFLP) analysis was one of the early methods to diagnose point mutations implicated in genetic diseases. This method was based on the observation that if a point mutation changes a restriction fragment recognition sequence, it is possible to detect this change by Southern blotting analysis in which the affected restriction enzyme is used to cut genomic DNA before analysis. 

                         The change in the size of detected fragments with a gene-specific probe signals the presence of mutation in the analyzed gene. For example, this method has been applied to the diagnosis of hemophilia A, which is the most common inherited bleeding disorder, affecting approx 1 in 5,000 males worldwide. Hemophilia A is an X-linked, recessively inherited bleeding disorder that results from a deficiency of procoagulant factor VIII (FVIII). Affected males suffer from joint and muscle bleeds and easy bruising, the severity of which is closely correlated with the level of activity of coagulation factor VIII (FVIII:C) in their blood. 

                          Gitschier et al. demonstrated using Southern blotting that it was possible to diagnose the disease in 42% of affected families (10). Having identified the BclI polymorphism, X-linked inheritance was demonstrated in three generations of a Utah family. DNA from a family member was restricted with BclI, electrophoresed on a 0.8% agarose gel, and blotted on to nylon membrane and probe with a complementary sequence within the factor VIII gene labeled with radioactivity. Twelve bands were observed by autoradiography. Eleven hybridizing bands remained constant, and one varied in position at either approx 0.9 or 1.1 kb in size.