Technical Sheet

Sickle cell anemia is an autosomal recessive disease. A single base change (A to T) in the b globin chain causes the substitution of amino acid glutamine to valine; the 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, under conditions of low oxygen tension the red cells become grossly abnormal assuming a sickle shape leading to aggregation and hemolysis. Homozygous Hb S is a serious hemoglobinopathy found almost exclusively in the Black population. About 8% of American Blacks are carriers and about 0.2% are affected. Heterozygotes (sickle cell trait) are clinically normal, although their red cells will sickle when subjected to very low oxygen pressure in vitro.

DNA analysis for the sickle cell mutation is done by specific amplification of the DNA region spanning the mutation using polymerase chain reaction followed by enzymatic cleavage of the amplified product. Sickle cell mutation abolishes a restriction endonuclease site (Dde I). Electrophoretic resolution of the fragment pattern reveals the presence or absence of the mutation. Clear genotyping of normal, carrier and homozygous DNA is achieved.

Reconstitution
1. Stock solution: Add 50m l sterile water to each tube containing the primer. The 10 nmoles of primer when dissolved in 50m l water will give a solution of 200 m Molar i.e. 200 pmoles/m l.
2. Primer Mix: Prepare a 10 pmoles/m l Primer Mix solution containing BOTH the primers. Example; add 180 m l water to a new tube. To this tube add 10 m l of EACH of the primer stock solution from #1 step above. Label this tube as
SC Primer Mix 10 pmoles/m l

30 cycles, 7 min. 72oC extension, 4oC soak.

B. Restriction enzyme digestion (100 m l reaction)
DNA 45 m l (45m l of 50 m l PCR reaction)
10 x buffer 10 m l
Dde I 10-30 units
Sterile water up to 100 m l

Precipitate after over night digestion, dissolve pellets in 5m l 1 x loading buffer.

C. Electrophoresis
Load samples to 1.5% agarose gel. Run at 90 mAmps for 2.5 hrs.

D. Results
Mutation abolishes restriction site.

Figure 1. Typical Sickle cell genotype analysis of PCR product digested with Dde I. Lane 1 is molecular weight markers. Lane 2 is undigested PCR product. Lanes 3, 4 and 6 is DNA with A/S geneotype. Lane 5 is A/A genotype DNA and Lane 7 represents DNA with S/S genotype.

**The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.