Direct Identification Of Sickle Cell Anemia Using Blot-Hybridization

Sickle cell anemia is an autosomal recessive disease that occurs as a result of a single base change (i.e., GAG to GTG) in the beta-globin chain that causes the substitution of amino acid glutamine to valine which is the chief cause of the disorder sickle cell anemia. The resulting mutant globin chain is termed as the Hb S. Hemoglobin S is freely soluble when fully oxygenated, however, under conditions of low oxygen tension, the red cells become abnormal taking a sickle shape leading to aggregation and hemolysis. Homozygous Hb S is a serious hemoglobinopathy found almost exclusively in the Black population.

Polymorphisms found in the human hemoglobin genes can be used as a means for the prenatal diagnosis of sickle cell anemia. The use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures provides a direct analysis. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5′-specific 13-globin gene probe, sickle cell trait individuals (AS) exhibit an additional band (376 base pairs) and patients with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. These changes in the banding pattern are supposed to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because analysis is performed in 20 micrograms of cellular DNA, the application to prenatal diagnosis of sickle cell anemia is possible.

DNA analysis for the sickle cell mutation is done by amplification of specific DNA region specifically the mutated DNA using polymerase chain reaction to amplify the gene followed by enzymatic cleavage of the amplified product. Direct analysis of the sickle cell anemia should be possible by the use of a restriction enzyme whose recognition sequence is created or eliminated by the sickle cell mutation. Sickle cell mutation destroys a restriction endonuclease site (Dde I). Electrophoretic resolution of the fragment pattern reveals the presence or absence of the mutation.

The method in brief for prenatal diagnosis of sickle cell disease: Clear genotyping of DNA is achieved by collecting blood samples from patients with sickle cell anemia, sickle cell trait, and control individuals with no known hematological disorder. DNA is then isolated and collected by ethanol precipitation and by repeated cycles of centrifugation at different speeds. The sample obtained is then treated with the restriction enzymes digestion and run on gel electrophoresis to isolate the individual fragment bands. DBM transfer, hybridization, and detection of sequences in the cellular DNA are done to see the mutated base.

The result obtained shows that the A to T transversion within the beta-globin gene sequence affects the restriction endonuclease recognition site for both enzymes Mnl I (G-A-G-G) and Dde I (C-T-N-A-G).

In conclusion, recombinant DNA techniques coupled with blot hybridization analysis have proven to be valuable tools for studying the molecular basis of hemoglobinopathies.