A microtiter plate assay for determining apolipoprotein E genotype and discovery of a rare allele

Genotype determination using the solid-phase minisequencing method of Syvänen et al. (1990, 1993) has been adapted for use with fluorescein-labeled dideoxynucleotides (F-ddNTPs). PCR is performed using one biotinylated primer and one unbiotinylated primer. The biotinylated products are captured in streptavidin-coated microtiter wells. Following removal of nonbiotinylated strands with NaOH, the bound strands are hybridized with a primer adjacent to the polymorphic site being tested. Using T7 DNA polymerase, the primer is extended using one F-ddNTP in the presence of the other three unlabeled ddNTPs. Incorporation of the F-ddNTP is detected by binding antifluorescein antibody conjugated with alkaline phosphatase followed by incubation with a chromogenic substrate. This assay was used to determine APOE genotypes for 75 subjects. The APOE genotypes were also determined using a method involving the incorporation of mobility-shifting nucleotide analogs (Livak et al., 1992). Investigation of the one discrepancy between the two methods revealed that one subject carries a rare APOE allele that has a 3 bp deletion.