DNA diagnosis by microchip capillary electrophoresis

微芯片毛细管电泳 DNA 诊断

基本信息

批准号：
12672252
负责人：
ARAKAWA Hidetoshi
金额：
$ 2.5万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672252/
关键词：
micro chip electrophoresis microchip capillary electrophoresis vero toxin S. mutans S. sobrinus PCR う食細菌 Duplex PCR 遺伝子診断 S.mutanns S.sobrinus SSCP 腸管出血性大腸菌

项目摘要

Gene diagnosis is utilized as diagnoses such as hereditary disease and infectious disease, and as diagnoses method such as the prediction of drug metabolic ability. Generally the molecular biological technique utilizing in the laboratory is used as DNA analysis of gene diagnosis. However, the rapid and high sensitive DNA analysis has been required in infectious disease and tailor made medicine. Recently, as the method, DNA chip method is developed, and the use in the clinical field is being tried. In this study, microchip electrophoresis was developed as high sensitive and rapid DNA analytical method, and the establishment of gene diagnosis which analyze DNA polymorphism with the one point mutation, O157 ( intestinal hemorrhagic Escherichia coli ) and mutans streptococcus was carried out. Construction of micro chip and selection of the polymer which is used as the separation medium were carried out. As the result, it was possible to analyze the PCR product of the O157 verotoxin gene ( … More VT1 and VT2 ) using the channel of 30 μm depth and 100 μm width at separation length 1mm in slight10 seconds. And, in order to detect the one point mutation in the ras gene which is one of the oncogenes, the PCR was done mutation site, and next, SSCP (single-strand conformation polymorphism) analysis was carried out. In this study, separation media such as polymer and gel were mainly examined. As the result, the analysis of single nucleotide polymorphism (SNP) was difficult, though it was able to separate in the difference of large loop structure. Next, DNA diagnosis method of mutans streptococcus which existed in dental plaque, was developed as a diagnosis method of the dental treatment and care. Duplex allele specific PCR and common PCR for S. mutans and S. sobrinus were developed. And the obtained PCR products were analyzed by the imcrochip electrophoresis. By this method, these genes could be analyzed in 3 minutes. And, the detection limit was high sensitive of 20 fg. By the research of the above, the microchip electrophoresis seems to be the usefulness as a DNA detection method of the infectious disease which requires speedy DNA analysis. Less

基因诊断被用作诊断，例如遗传性疾病和传染病，以及诊断方法，例如预测药物代谢能力。通常，利用实验室中利用的分子生物学技术用作基因诊断的DNA分析。但是，在传染病和量身定制的药物中需要快速且高度敏感的DNA分析。最近，随着该方法，开发了DNA芯片方法，并尝试了在临床领域的使用。在这项研究中，微芯片电泳是作为高灵敏和快速DNA分析方法开发的，并且基因诊断的建立可以通过一个点突变O157（肠胃出血性埃斯氏菌大肠杆菌）和MUTANS链球菌分析DNA多态性。进行微芯片的构造和选择用作分离培养基的聚合物的选择。结果，可以使用30μm深度和100μm宽度的通道在稍有10秒内分离长度为1mm，可以分析O157 Verotoxin基因（…更多VT1和VT2）的PCR产物。并且，为了检测Ras基因中的一个点突变，这是癌基因的一种突变，进行了PCR突变位点，然后进行了SSCP（单链构象多态性）分析。在这项研究中，主要检查了分离培养基，例如聚合物和凝胶。结果，很难分析单个核苷酸多态性（SNP），尽管它能够在大环结构的差异上分离。接下来，牙菌斑中存在的链球菌的DNA诊断方法是作为牙科治疗和护理的诊断方法开发的。开发了双链等位基因的特异性PCR和用于突变链球菌和S. sobrinus的普通PCR。并通过Imcrochip电泳分析获得的PCR产物。通过这种方法，可以在3分钟内分析这些基因。并且，检测极限高度敏感为20 fg。通过上述研究，微芯片电泳似乎是传染病的DNA检测方法的有用性，需要快速的DNA分析。较少的