Abstract
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., N. Wood, and S. E. Humphries. Rapid determination of apolipoprotein E genotype using a heteroduplex generator.
Highlights
The apoE gene exhibits two common polymorphisms that have been associated with both coronary artery disease and Alzheimer’s disease
There was no need to re-check any samples typed using heteroduplex analysis whilst several of those samples typed using HhaI had to be re-analyzed mainly due to poor amplification resulting in faint bands
Heteroduplex analysis has been shown to be a simple and rapid method for the identification of mutations [31,32,33,34,35, 37, 38], involving the amplification of the mutated and normal genomic DNA followed by non-denaturing polyacrylamide gel electrophoresis
Summary
The apoE gene exhibits two common polymorphisms that have been associated with both coronary artery disease and Alzheimer’s disease. Numerous methods have been described to identify these three apoE alleles there are disadvantages and ambiguities associated with all of them. 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 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.
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