Bolla M K, Haddad L, Humphries S E, Winder A F, Day I N
Department of Medicine, Rayne Institute, University College of London Medical School, UK.
Clin Chem. 1995 Nov;41(11):1599-604.
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.
分子流行病学研究已经确定了一种常见的载脂蛋白E（apo E）异构体（与E3相对的E4）与冠状动脉疾病和阿尔茨海默病痴呆风险之间的关联。此外，apo E基因型（通常为E2/E2）在Ⅲ型高脂血症中的作用是众所周知的。然而，无论是出于诊断还是研究目的，apo E基因分型都很繁琐。首选方法是对限制性消化片段进行电泳大小分析，从而能够同时分析代表六种常见基因型（E2/E2；E2/E3；E2/E4；E3/E3；E3/E4；E4/E4）的两个密码子（112和158）。然而，随之而来的对高产率PCR、高分辨率、高通量电泳以及足够检测灵敏度的要求，使得已发表的方案存在不足。结合我们最近描述的一种高通量电泳系统——微孔板阵列对角凝胶电泳（MADGE），我们构建了经过广泛优化和简化的方案，用于从漱口样本中分离DNA以进行PCR设置和高产率PCR、限制性消化以及随后的MADGE凝胶图像分析。该集成系统使一名工作人员无需特殊设备就能轻松地每天对多达数百个DNA样本进行apo E基因分型。
