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. © 1994 Wiley‐Liss, Inc.