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.
Livak
K J
KJ
Research & Development Division, Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19801.
Hainer
J W
JW
D000483:Alleles; D001057:Apolipoproteins E / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D017931:DNA Primers; D003854:Deoxyribonucleotides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005452:Fluoresceins; D015200:Fluoroimmunoassay / Q000379:methods; D005838:Genotype; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin; D017186:Titrimetry / Q000379:methods
D001057:Apolipoproteins E; D001426:Bacterial Proteins; D017931:DNA Primers; D003854:Deoxyribonucleotides; D005452:Fluoresceins; D001710:Biotin; D019809:Streptavidin