Open AccessApolipoprotein e genotyping methods for the clinical laboratory
Author(s) -
Maekawa Baku,
Cole Thomas G.,
Seip Richard L.,
Bylund David
Publication year - 1995
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1860090112
Subject(s) - microbiology and biotechnology , restriction enzyme , genotyping , genotype , oligonucleotide , chemistry , polymerase chain reaction , restriction fragment , dna , gel electrophoresis , polyacrylamide gel electrophoresis , endonuclease , biology , biochemistry , gene , enzyme
To select the best method for detecting apolipoprotein E (apo E) genotypes determined by the three common alleles ϵ2, ϵ3 and ϵ4, 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 Hha I, 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.©1995 wiley‐Liss, inc.