Rapid determination of apolipoprotein E genotype using a heteroduplex generator.
The apoE gene exhibits two common polymorphisms that have been associated with both coronary artery disease and Alzheimer's disease. The polymorphisms create the three allelic isoforms E2, E3, and E4 which are encoded by Cys;-Cys, Cys;-Arg, and Arg;-Arg at amino acid positions 112 and 158, respectively. Numerous methods have been described to identify these three apoE alleles although there are disadvantages and ambiguities associated with all of them. Here we describe a method by which the two common apoE polymorphisms can be identified simultaneously. The method involves PCR of the region containing the two polymorphic sites, followed by hybridization of this PCR product to a synthetic molecule called a universal heteroduplex generator (UHG). The UHG is used to induce heteroduplex formation which is visualized on a non-denaturing mini-gel using ethidium bromide staining. This technique which can also identify other rare mutations in the amplified region of DNA under investigation, is an unequivocal method of genotyping and is simpler and faster than many methods, including using restriction enzyme digestion.
Bolla
M K
MK
Centre for Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School, The Rayne Institute, 5 University Street, London, WC1E 6JJ, UK.
Wood
N
N
Humphries
S E
SE
D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011110:Polymorphism, Genetic; D020033:Protein Isoforms; D015203:Reproducibility of Results
D001057:Apolipoproteins E; D020033:Protein Isoforms