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、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)检测随机位置点突变的新方法。对于DNA提取方法，我们比较了包括商业玻璃珠法在内的4种方法。我们的结果表明，经过细微修改的玻璃珠方法非常简单且有效，并且获得的 DNA 样品适合 PCR 分析。为了防止残留污染，我们测试了尿酰 N-糖基化酶系统。经证实，dUTP-尿酰基N-糖基化酶系统可以是防止在未专门配备用于PCR研究的常规实验室中进行的PCR中残留污染的唯一实用方法。我们尝试使用非放射性同位素探针从临床样本中检测 Ki-ras 基因的点突变。化学发光系统被证明适合此目的，因为其他染色方法会导致高背景信号。我们还尝试建立一种新方法来检测随机位置发生的点突变。第一个方案是基于S1核酸酶对野生型和突变基因杂交后形成的异源双链体进行特异性消化。结果表明，S1 核酸酶消化对于我们的目的来说不够具体。其他方法的试验正在进行中。