A MICROTITER PLATE ASSAY FOR DETERMINING APOLIPOPROTEIN-E GENOTYPE AND DISCOVERY OF A RARE ALLELE

Genotype determination using the solid-phase minisequencing method of Syvanen et al. (1990, 1993) has been adapted for use with fluorescein- labeled dideoxynucleotides (F-ddNTPs). PCR is performed using one biot inylated primer and one unbiotinylated primer. The biotinylated produc ts are captured in streptavidin-coated microtiter wells. Following rem oval of nonbiotinylated strands with NaOH, the bound strands are hybri dized with a primer adjacent to the polymorphic site being tested. Usi ng T7 DNA polymerase, the primer is extended using one F-ddNTP in the presence of the other three unlabeled ddNTPs. Incorporation of the F-d dNTP is detected by binding antifluorescein antibody conjugated with a lkaline phosphatase followed by incubation with a chromogenic substrat e. This assay was used to determine APOE genotypes for 75 subjects. Th e APOE genotypes were also determined using a method involving the inc orporation of mobility-shifting nucleotide analogs (Livak et al., 1992 ). Investigation of the one discrepancy between the two methods reveal ed that one subject carries a rare APOE allele that has a 3 bp deletio n. (C) 1994 Wiley-Liss, Inc.