Development of High-Performance Gene Diagnosis System for Common Diseases Using Capillary Electrophoresis

许多人类疾病是由致病基因的DNA序列的微小变化引起的。DNA序列的改变包括致病基因的多种类型的突变和多态性，如一个或多个核苷酸的替换、某个序列的缺失或插入、可变数目的串联重复序列(VNTR)座位的差异，以及微卫星重复序列的不稳定性。因此，人类疾病的DNA诊断是通过分析致病基因的DNA序列变化来实现的。在这项研究中，我们开发了多色可检测的激光诱导荧光(LIF)检测系统，用于毛细管电泳法和毛细管阵列电泳法，并检测了该系统与聚合酶链式反应(PCR)技术相结合对致病基因进行快速基因分型和人类疾病的高速DNA诊断的效率和性能。该体系成功地应用于载脂蛋白E基因(APOE)和p53抑癌基因的快速基因分型，通过对载脂蛋白B基因(APOB)的VNTR位点进行分型检测载脂蛋白B基因(APOB)，通过微卫星不稳定性分析检测DNA错配修复基因。此外，该系统还成功地应用于癌症(P53和DNA错配修复基因)、阿尔茨海默病(APOE)、冠心病(APOB)和强直性肌营养不良(三重重复)的DNA诊断。在这些情况下，这里显示的许多数据表明，毛细管电泳联用聚合酶链式反应技术是一种高通量、高准确度、低劳动强度和低成本的人类疾病DNA诊断技术。

Many human diseases are caused by small alterations in DNA sequence of the disease-causing genes. The alterations in DNA sequence include many types of mutation and polymorphism on disease-causing genes, such as one or several nucleotides replacement, deletion or insertion of some sequence, differences in a variable number of tandem repeat (VNTR) locus, and the instability of microsatellite repeat. DNA diagnosis for human diseases is, therefore, realized by the analysis of the alterations in the DNA sequence of disease-causing genes. In this study, we developed multicolor detectable laser-induced fluorescence (LIF) detection system for capillary electrophoresis and capillary array electrophoresis, and examined the efficiency and the performance of the systems coupled with polymerase chain reaction (PCR) technique for rapid genotyping of disease-causing genes and high-speed DNA diagnosis for human diseases. The system is successfully applied to rapid genotyping of apolipoprotein E gene (APOE) and p53 tumor suppressor gene through detection of one nucleotide replacement, apolipoprotein B gene (APOB) through typing of its VNTR locus, detection of an expansion for triplet repeat, and DNA mismatch repair gene through microsatellite instability analysis. Additionally, the systems are successfully applied to DNA diagnosis for cancer (p53 and DNA mismatch repair gene), Alzheimer's disease (APOE), coronary heart disease (APOB), and myotonic dystrophy (triplet repeat). In these instances, many data shown here demonstrate that capillary electrophoresis coupled with the PCR technique is a promising technology for DNA diagnosis of human diseases with high throughput, high accuracy, less labor-intensive, and cost effectiveness.