Development of High-Performance Gene Diagnosis System for Human Diseases Using Integrated Microdevice Technology

利用集成微器件技术开发高性能人类疾病基因诊断系统

• 批准号: 09557190
• 负责人: BABA Yoshinobu
• 金额: $ 7.94万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557190/
• 关键词: Microdevice; Microchip; Gene Diagnosis; PCR

Many human diseases are caused by small alterations in DNA sequence of the disease-causing genes. 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 microfabricated device integrated by PCR and capillary array electrophoresis on a chip. Laser-induced fluorescence detection system for the chip has been constructed by the modification of laser confocal fluorescence microscopy and we investigated DNA conformational dynamics using fluorescence microscopy. Even sigle DNA molecule labeled by intercalating fluorescence dyes was detected during migrating into the array of microchannels and conformational dynamics of single DNA molecule in microchannels filled with linear polyacrylamide under electric field was observed by this system to elucidate the separation mechanism of DNA and to optimize the separation conditions for DNA analysis. In the optimum conditions established in this study, DNA fragments up to 1 kbp were separated within only 60 s. In these instances, many data shown here demonstrate that integrated microdevice technology 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。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。在本研究中，我们开发了一种集成了PCR和毛细管阵列电泳的芯片微加工装置。通过对激光共聚焦荧光显微镜的改进，构建了用于该芯片的激光诱导荧光检测系统，并利用荧光显微镜研究了DNA的构象动力学。利用该系统可以检测到嵌入荧光染料标记的单个DNA分子在微通道阵列中的迁移，并观察了电场作用下单个DNA分子在线性聚丙烯酰胺填充的微通道中的构象动力学，以阐明DNA的分离机理，优化DNA分析的分离条件。在本研究中建立的最佳条件下，DNA片段高达1 kbp的分离时间仅为60 s。在这些情况下，这里显示的许多数据表明，集成微器件技术是一种很有前途的技术，用于人类疾病的DNA诊断，具有高通量，高精度，劳动密集度低，成本效益高。

项目成果

Y.Baba: "Simultaneous Genotyping of Cholesteryl-Ester Transfer Protein Gene by Duplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis Using CE" J.Chromatogr.B. (発表予定). (1999)

Y.Baba：“使用 CE 通过双链聚合酶链反应限制性片段长度多态性分析对胆固醇酯转移蛋白基因进行同步基因分型”J.Chromatogr.B（待提交）。

Y.Baba: "DNA Sequencing and Gene Diagnosis by Capillary Electrophoresis" Igakuno Ayumi. 184 (4). 285-289 (1997)

Y.Baba：“通过毛细管电泳进行 DNA 测序和基因诊断”Igakuno Ayumi。

Y.Kiba: "Capillary Electrophoretic Behavior of Triplet Repeat DNA and Formation of Specific Higher-Ordered Structure" SeibutsuButsuri. 38 (6). 253-255 (1998)

Y.Kiba：“三联体重复 DNA 的毛细管电泳行为和特定高阶结构的形成”SeibutsuButsuri。

Y.Baba: "Genomic and Proteomic Drug Discovery" Eur.J.Pharm.Sci.(in press). (1999)

Y.Baba：“基因组和蛋白质组药物发现”Eur.J.Pharm.Sci.（印刷中）。

木村彰: "「医療におけるバイオテクノロジー」第2章3節" 医薬ジャーナル(発表予定),

Akira Kimura：“‘医疗保健中的生物技术’第 2 章第 3 节”Pharmaceutical Journal（待出版），