Gioia L, Vogt L J, Freeman W M, Flood A, Vogt B A, Vrana K E
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1083, USA.
J Neurosci Methods. 1998 Apr 30;80(2):209-14. doi: 10.1016/s0165-0270(98)00002-8.
A technique is described for determining the apolipoprotein E genotype (apo E; alleles epsilon2, epsilon3, or epsilon4) from tissues which have been fixed with 4-10% formaldehyde and archived. The procedure requires efficient extraction and exhaustive purification of DNA from the fixed tissue. Because the fixation process renders the DNA largely crosslinked and/or sheared (therefore unsuitable for traditional analysis), a nested polymerase chain reaction (PCR) is employed (using two apo E gene specific primer pairs) to specifically amplify the polymorphic region of the gene. The genotype was then determined using previously reported HhaI polymorphisms that occur as a direct result of the variant codons responsible for the three alleles. This protocol permitted the successful genotyping of 90% (34 out of 38) of the archived brain samples from Alzheimer's disease (AD) patients. These samples included such extremes as a sample that had been stored for 12 years in formalin. This procedure permits the retrospective analysis of samples that had been processed and stored well before the original characterization of apo E alleles as risk factors in AD. Finally, this approach is readily adapted to the analysis of any gene of interest, whether by restriction fragment length polymorphism or direct amplicon DNA sequencing. It is also a very robust assay for less stringent conditions such as DNA isolated from whole blood or frozen tissue.
本文描述了一种从用4-10%甲醛固定并存档的组织中确定载脂蛋白E基因型（apo E；ε2、ε3或ε4等位基因）的技术。该方法需要从固定组织中高效提取和彻底纯化DNA。由于固定过程使DNA大量交联和/或剪切（因此不适合传统分析），所以采用巢式聚合酶链反应（PCR）（使用两对apo E基因特异性引物）来特异性扩增该基因的多态性区域。然后使用先前报道的由导致三个等位基因的变异密码子直接产生的HhaI多态性来确定基因型。该方案成功地对90%（38个中的34个）来自阿尔茨海默病（AD）患者的存档脑样本进行了基因分型。这些样本包括在福尔马林中储存了12年的样本等极端情况。该方法允许对在apo E等位基因最初被鉴定为AD风险因素之前就已处理和储存的样本进行回顾性分析。最后，这种方法很容易适用于对任何感兴趣基因的分析，无论是通过限制性片段长度多态性还是直接扩增子DNA测序。对于不太严格的条件，如从全血或冷冻组织中分离的DNA，这也是一种非常可靠的检测方法。
