Optimization of apolipoprotein E genotyping
Optimization of apolipoprotein E genotyping
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DOI：
10.1016/s1084-8592(97)80038-0
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Leonard, DGB
中科院分区：
文献类型：
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作者：
Addya, K;Wang, YL;Leonard, DGB
背景：载脂蛋白 E (apoE) 的三个常见等位基因已被鉴定，并共显性表达以产生六种基因型。不同的 apoE 基因型与多种心血管和神经系统疾病有关。 apoE 基因型检测的诊断重要性日益增加，特别是在冠状动脉疾病的风险评估中。开发了一种可重复且经济高效的检测方法。方法和结果：在二甲亚砜存在下，使用两步热循环对 apoE 基因第四外显子进行聚合酶链式反应 (PCR) 扩增。 PCR产物用HhaI限制性内切酶消化并通过琼脂糖凝胶电泳分析以确定apoE基因型。已确定并优化了包括二甲基亚砜、DNA 浓度和 PCR 循环条件等多种因素对 PCR 特异性和效率的影响。结论：通过 PCR 限制性片段长度多态性分析进行载脂蛋白 E 基因分型已针对临床诊断实验室的使用进行了优化，允许一名技术人员一天内评估多达 52 个样本。
Background: Three common alleles of apolipoprotein E (apoE) have been identified and are expressed codominantly to generate six genotypes. Different apoE genotypes are implicated in several cardiovascular and neurologic disorders. Testing for apoE genotypes has increasing diagnostic importance, particularly in the risk assessment of coronary artery disease. A reproducible and cost-effective assay was developed.Methods and Results: Polymerase chain reaction (PCR) amplification of the fourth exon of the apoE gene is performed in the presence of dimethyl sulfoxide using two-step thermal cycling. The PCR products are digested with HhaI restriction enzyme and analyzed by agarose gel electrophoresis to determine apoE genotypes. Effects of several factors, including dimethyl sulfoxide, DNA concentration, and PCR cycling conditions, on PCR specificity and efficiency have been determined and optimized.Conclusions: Apolipoprotein E genotyping by a PCR restriction fragment length polymorphism analysis has been optimized for use in a clinical diagnostic laboratory, allowing evaluation of up to 52 samples by one technician in one day.