Optimization of Apolipoprotein E Genotyping.
Background: Three common alleles of apolipoprotein E (apoE) have been identified and are expressed codominantly to generate six genotypes. Different apoE genotypes are implicated in several cardiovascular and neurologic disorders. Testing for apoE genotypes has increasing diagnostic importance, particularly in the risk assessment of coronary artery disease. A reproducible and cost-effective assay was developed. Methods and Results: Polymerase chain reaction (PCR) amplification of the fourth exon of the apoE gene is performed in the presence of dimethyl sulfoxide using two-step thermal cycling. The PCR products are digested with HhaI restriction enzyme and analyzed by agarose gel electrophoresis to determine apoE genotypes. Effects of several factors, including dimethyl sulfoxide, DNA concentration, and PCR cycling conditions, on PCR specificity and efficiency have been determined and optimized. Conclusions: Apolipoprotein E genotyping by a PCR restriction fragment length polymorphism analysis has been optimized for use in a clinical diagnostic laboratory, allowing evaluation of up to 52 samples by one technician in one day.
Addya
K
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA
Wang
YL
Leonard
DG