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

本课题主要研究了以下几点：（1）从临床标本中提取DNA的简易方法。（2）防止PCR产物的携带污染。（3）用非放射性同位素探针检测点突变。（4）检测随机位点突变的新方法。在DNA提取方法上，我们比较了包括商业玻璃珠法在内的4种方法。结果表明，玻璃微珠法操作简单，效率高，所得DNA样品可用于PCR分析。为了防止携带污染，我们测试了尿嘧啶N-糖基化酶系统。已证实dUTP-尿嘧啶N-糖基化酶系统是在没有专门配备PCR研究设备的常规实验室中进行PCR时防止残留污染的唯一实用方法。我们试图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用于此目的，因为其他染色方法导致高背景信号。我们还尝试建立一种新的方法来检测点突变发生在一个随机的位置。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的特异性消化。结果表明，S1核酸酶消化对我们的目的不够特异。其他方法的试验正在进行中。

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.