Rapid determination of apolipoprotein E genotype using a heteroduplex generator

Abstract

The apoE gene exhibits two common polymorphisms that have been associated with both coronary artery disease and Alzheimer's disease. The polymorphisms create the three allelic isoforms E2, E3, and E4 which are encoded by Cys-Cys, Cys-Arg, and Arg-Arg at amino acid positions 112 and 158, respectively. Numerous methods have been described to identify these three apoE alleles although there are disadvantages and ambiguities associated with all of them. Here we describe a method by which the two common apoE polymorphisms can be identified simultaneously. The method involves PCR of the region containing the two polymorphic sites, followed by hybridization of this PCR product to a synthetic molecule called a universal heteroduplex generator (UHG). The UHG is used to induce heteroduplex formation which is visualized on a non-denaturing mini-gel using ethidium bromide staining. This technique which can also identify other rare mutations in the amplified region of DNA under investigation, is an unequivocal method of genotyping and is simpler and faster than many methods, including using restriction enzyme digestion. -Bolla, M. K., N. Wood, and S. E. Humphries. Rapid determination of apolipoprotein E genotype using a heteroduplex generator.

Plasma proteins, cognitive decline, and 20‐year risk of dementia in the Whitehall II and Atherosclerosis Risk in Communities studies

“…Experienced clinical nurses measured body mass index (BMI) and systolic blood pressure and took blood samples for lipid and glucose measurements 19 . Using DNA extracted from whole blood, a standard polymerase chain reaction (PCR) assay determined apolipoprotein E ( APOE ) genotype using the salting out method 34,35 . Two blinded independent observers read the genotype and any discrepancies were resolved by repeating the PCR analysis.…”

“… 19 Using DNA extracted from whole blood, a standard polymerase chain reaction (PCR) assay determined apolipoprotein E ( APOE ) genotype using the salting out method. 34 , 35 Two blinded independent observers read the genotype and any discrepancies were resolved by repeating the PCR analysis. In ARIC, baseline covariates included age, sex, and a combined race–study center variable, measured using standard operating protocols.…”

Immune system-wide Mendelian randomization and triangulation analyses support autoimmunity as a modifiable component in dementia-causing diseases

“…In the Whitehall II study, standard self-administered questionnaires provided data on age and sex. Using DNA extracted from whole blood, a standard PCR assay determined APOE genotype using the salting-out method 110 , 111 . Two blinded independent observers read the genotype, and any discrepancies were resolved by repeating the PCR analysis.…”

Immune system and blood-brain barrier-wide biomarker analyses provide causal evidence for autoimmunity in dementia

“…Using DNA extracted from whole blood, a standard PCR assay determined APOE genotype using the salting out method. 103, 104 In analyses, proteins were transformed to a normal distribution by inverse rank-based normal transformation. The analyses used plasma samples measured in 1995/1997 and the follow-up ended at onset of dementia, death, or 1 st of October 2019, which ever occurred first.…”

“…Using DNA extracted from whole blood, a standard PCR assay determined APOE genotype using the salting out method. 20, 21 Two blinded independent observers read the genotype and any discrepancies were resolved by repeating the PCR analysis.…”