Detection of apolipoprotein E genotypes by capillary electrophoresis

Abstract The apolipoprotein E (apo-E) genotype of an individual is of significant relevance in the associated risk of developing cardiovascular disease and late-onset Alzheimer's disease. Detection of the six common apo-E genotypes is based on the restriction fragment length polymorphisms (RFLPs) arising from the abolition or creation of Hha I restriction sites within an amplified target DNA sequence of the apo-E gene. Genomic DNA was extracted from leukocytes, a 230 bp target sequence within the apo-E gene was amplified by polymerase chain reaction (PCR) and digested with Hha I, and the restricted DNA fragments separated by capillary electrophoresis (CE). This was performed on the BioFocus™ 3000 automated CE system equipped with an experimental laser-induced fluorescence (LIF) detector (Bio-Rad Laboratories, Hercules, CA), using capillaries (27 cm length, 75 μm i.d.) coated internally with polyaminoacryloylethoxyethanol. The analysis buffer (2×Tris borate–EDTA, pH 8.3) was supplemented with a proprietary sieving polymer and 0.05 μM thiazole orange six. Samples were injected electrophoretically. Separations were carried out at 40°C under constant voltage, and the emitted fluorescence detected at 515 nm. Restriction fragment lengths of the cleaved PCR products were estimated from the migration times, with a 20/100 bp ladder (Bio-Rad Laboratories 20/100 bp molecular ruler) serving as reference. Six different reproducible patterns were obtained for the six common apo-E genotypes, with good resolution of the component restriction fragments. The calculated sizes of the separated peaks closely corresponded with the predicted restricted fragment lengths for each specific genotype. We believe this is the first published report demonstrating the feasibility of automating the post-PCR detection of the apo-E RFLPs 2 . This methodology overcomes the most labour-intensive step in apo-E genotyping, thus making it amenable to routine clinical application.