High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis.
Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.
Bolla
M K
MK
Department of Medicine, Rayne Institute, University College of London Medical School, UK.
Haddad
L
L
Humphries
S E
SE
Winder
A F
AF
Day
I N
IN
D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004586:Electrophoresis / Q000379:methods*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012463:Saliva / Q000737:chemistry; D013698:Templates, Genetic
D001057:Apolipoproteins E; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases