title: Apolipoprotein E genotyping methods for the clinical laboratory.
abstract: To select the best method for detecting apolipoprotein E (apo E) genotypes determined by the three common alleles epsilon 2, epsilon 3 and epsilon 4, we compared the radiolabeled allele-specific oligonucleotide (ASO) probe assay and the nonisotopic restriction isotyping assay. Leukocytic DNA was extracted from the blood of 93 patients after which the region containing two mutation points coding amino acid residues 112 and 158 was amplified by using the polymerase chain reaction (PCR). Amplified DNA fragments were spotted on nylon membranes, then hybridized for the ASO probe assay. The amplified DNA fragments were also digested with restriction endonuclease Hhal, followed by polyacrylamide gel electrophoresis for the restriction isotyping assay. The apo E genotypes determined by both methods for every specimen studied were in complete agreement. Although the radiolabeled ASO probe method was 10 times more sensitive than restriction isotyping on polyacrylamide gel, the two were comparable in accuracy. Additionally, because it is simpler to perform, is less time consuming, and is less expensive, we conclude that the restriction isotyping assay is the more suitable of these two methods for use in a clinical laboratory.
authors.affiliation: Tsukuba Research Laboratory, Hitachi Chemical Co., Ltd., Ibaraki, Japan.
authors.forename: B
authors.initials: B
authors.lastname: Maekawa
authors.forename: T G
authors.initials: TG
authors.lastname: Cole
authors.forename: R L
authors.initials: RL
authors.lastname: Seip
authors.forename: D
authors.initials: D
authors.lastname: Bylund
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001057:Apolipoproteins E / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D005260:Female; D005838:Genotype; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping
chemical_list: D001057:Apolipoproteins E; D015345:Oligonucleotide Probes