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

高性能毛细管电泳常见疾病基因诊断系统的开发

• 批准号: 08672490
• 负责人: BABA Yoshinobu
• 金额: $ 1.34万
• 依托单位: The University of Tokushima (1997)Kobe Pharmaceutical University (1996)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672490/
• 关键词: Capillary Electrophoresis; PCR; Gene Dianosis; Cancer; Alzheimer's disease; Heart Disease

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.

许多人类疾病是由致病基因的DNA序列的微小改变引起的。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、某些序列的缺失或插入、可变数目串联重复序列（VNTR）位点的差异、微卫星重复序列的不稳定等。因此，人类疾病的DNA诊断是通过分析致病基因的DNA序列中的改变来实现的。本研究建立了毛细管电泳和毛细管阵列电泳的激光诱导荧光（LIF）检测系统，并将其与聚合酶链反应（PCR）技术相结合，研究了LIF检测系统在致病基因快速分型和人类疾病快速DNA诊断中的效率和性能。该系统成功地应用于载脂蛋白E基因（APOE）和p53肿瘤抑制基因（p53）的一个核苷酸替换检测、载脂蛋白B基因（APOB）的VNTR位点分型、三联体重复扩增检测和DNA错配修复基因的微卫星不稳定性分析。此外，该系统还成功应用于癌症（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（三联体重复）的DNA诊断。在这些情况下，这里显示的许多数据表明，毛细管电泳结合PCR技术是一种有前途的技术，用于人类疾病的DNA诊断，具有高通量，高准确性，劳动密集度低，成本效益。

项目成果

H.Kimura: Biotechnology in Medicine. Iyaku Journal (in press),

H.Kimura：医学生物技术。

Y.Baba: "CA Repeat Analysis by Capillary Electrophoresis in Non-denaturing Polymer Solutions." Analyst. (発表予定).

Y.Baba：“非变性聚合物溶液中的毛细管电泳 CA 重复分析。”（待介绍）。

M.Ueda: "Electrophoresis of Long DNA Molecules in Linear Polyacrylamide Solution" Biophys.Chem.(発表予定).

M.Ueda：“线性聚丙烯酰胺溶液中长 DNA 分子的电泳”Biophys.Chem。

馬場 嘉信: "電気泳動チップによるヒトゲノムの超高速解析" ファルマシア. 32・5. 528-529 (1996)

Yoshinobu Baba：“使用电泳芯片对人类基因组进行超高速分析” Pharmacia 32・529（1996）。

H.Inoue: "Enhanced Separation of DNA Sequencing Products by Capillary Electrophoresis Using Stepwise Gradient of Electric Field Strength" J.Chromatogr.A. (in press).

H.Inoue：“使用电场强度逐步梯度通过毛细管电泳增强 DNA 测序产品的分离”J.Chromatogr.A。