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

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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.