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Studies for developing a laboratory diagnostic system for cancers by means of PCR that is applicale to clinical samples such as urine and stool

研究开发适用于尿液和粪便等临床样本的 PCR 癌症实验室诊断系统

基本信息

批准号：
05557059
负责人：
WATANABE Toshiki
金额：
$ 10.05万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

In this project, we studied the following points : (1)Simple methods extracting DNA from clinical materials such as urine and stool. (2)Prevention of carry-over contamination of PCR products. (3)Detection of point mutaion by non-radioisotpoe probes. (4)New method for detecting point mutation ocurring at random position. As for the method of DNA extraction, we compared 4 methods including commercial glass beads method. Our results indicated that the glass beads method with a minor modification was very simple and worked efficiently, and the DNA sample obtained was good for PCR analysis. For preventing the carry-over contamination, we tested the Uracyl N-glycosylase system. It was confirmed that dUTP-Uracyl N-glycosylase system can be the only practical method to prevent carry-over contamination in the PCR performed in the conventional laboratories not specificaly equipped for PCR studies. We tried to detect a point mutation in the Ki-ras gene from clinical samples using non-radioisotope probes. Chemiluminescence system proved to be suitable for this purpose, because other staining method resulted in high background signals. We also tried to establish a new method for detecting a point mutaion ocurring at a random position. The first plan was based on the specific digestion by S1 nuclease of the heteroduplex formed by wild type and mutated genes after hybridization. It was revealed that S1 nuclease digestion was not specific enough for our purpose. Trials of other methods are now under way.

在本项目中，我们研究了以下几点：(1)从尿液和粪便等临床材料中提取DNA的简单方法。(2)防止PCR产物的残留污染。(3)非放射性同位素探针点突变检测。(4)检测发生在任意位置的点突变的新方法。对于DNA的提取方法，比较了市售玻璃微珠法等4种方法。结果表明，玻璃微珠法操作简单，操作效率高，所获得的DNA样品能很好地用于PCR分析。为了防止残留污染，我们测试了尿酰N-糖基酶系统。已证实，在常规实验室进行的聚合酶扩增中，dUTP-尿酰N-糖基酶体系是防止残留污染的唯一实用方法，而不是专门用于聚合酶链式反应研究的。我们试图使用非放射性同位素探针从临床样本中检测Ki-ras基因点突变。化学发光体系被证明是适合于这一目的的，因为其他染色方法会产生高背景信号。我们还试图建立一种新的方法来检测在任意位置发生的点突变。第一个方案是基于S1DNA酶对野生型基因和突变基因杂交后形成的异源双链的特异性消化。结果表明，S1核酸酶消化的特异性不足以满足我们的要求。其他方法的试验目前正在进行中。