Schlenck A, Bohnet K, Aguillon D, Lafaurie C, Siest G, Visvikis S
Centre de Médecine Préventive, Nancy, France.
Biotechniques. 1997 Apr;22(4):736-42. doi: 10.2144/97224rr03.
Human apolipoprotein E (apoE) is a product of a polymorphic gene. In the general population, it shows two major mutations, which lead to the appearance of three common alleles encoding for three protein isoforms. This polymorphism is important in the regulation of lipid metabolism. Accurate apoE phenotyping or genotyping has become essential in clinical laboratories, since the epsilon 4 allele has been associated with cardiovascular and Alzheimer's diseases. Endonuclease restriction isotyping, followed by slab gel electrophoresis, is a rapid and convenient method for the investigation of common apoE genotypes. However, during the large-scale apoE genotyping of the STANISLAS cohort, we were confronted with a partial lack of sensitivity and resolution power of this traditional method, which sometimes leads to the misclassification of the genotypes epsilon 2/2 and epsilon 3/2. We have overcome this difficulty by separating the restriction fragments with capillary gel electrophoresis linked to laser-induced fluorescence detection. The baseline resolution was 2 bp, and the sensitivity limit attainable was similar to that by radioactive detection. The distinction between the epsilon 3/2 and the epsilon 2/2 genotypes became unequivocal, even when only low amounts of DNA were available for amplification.
人类载脂蛋白E（apoE）是一个多态性基因的产物。在普通人群中，它表现出两种主要突变，这导致出现编码三种蛋白质异构体的三个常见等位基因。这种多态性在脂质代谢调节中很重要。准确的apoE表型分型或基因分型在临床实验室已变得至关重要，因为ε4等位基因与心血管疾病和阿尔茨海默病有关。内切酶限制性同型分型法，随后进行平板凝胶电泳，是一种快速便捷的研究常见apoE基因型的方法。然而，在对斯坦尼斯拉斯队列进行大规模apoE基因分型时，我们遇到了这种传统方法部分缺乏敏感性和分辨力的问题，这有时会导致ε2/2和ε3/2基因型的错误分类。我们通过使用与激光诱导荧光检测相连的毛细管凝胶电泳分离限制性片段克服了这一困难。基线分辨率为2 bp，可达到的灵敏度极限与放射性检测相似。即使只有少量DNA可用于扩增，ε3/2和ε2/2基因型之间的区分也变得明确无误。
