Study Of Single-strand Probes RDPCR And The DNA Damage Of Rats P53 Gene

Many studies indicated there was signify relationship between DNA damage and the happen and develop of many tumors. DNA damage is a detectable key step during the environmental carcinogenesis and its detection has very important toxicological significance. The randomized terminal linker-Dependent PCR (RDPCR), the newly innovated method by our laboratory, is established on the base of the ligation-mediated polymerase chain reaction (LMPCR) technique which is established by Pfeifer et al. can detect DNA damage. Some important results have been obtained by use of RDPCR. But during the course of experimentation, we found the sensibility and the specificity of this technology were not very good .Some data testified the sensibility and the specificity of single-strand probes(ss probes) were better than double-strand probes(ds probes), so using ss probes insteaded ds probes perhans will be helpful for solve the problem.In this study, we plan to prepare ss probes using single-primer PCR and asymmetric PCR,compare the results hybridized with ss probes and ds probes,and apply ss probes in RDPCR in vivo to detect DNA damage inducedby several environmental carcinogens. We hope to improve the sensibility and the specificity of RDPCR by applying ss probes, reduce false negative and false positive, perfect this technology, explored the possible molecular mechanism of mutagenesis/ carcinogenesis and expand the appliance range of RDPCR.Part I Study of ss probes RDPCR§ 1.Preparation of ss probesIn this part , the 187bp segment(double strand) of the exon7 of the p53 gene was successfully amplified by general PCR in which the genomic DNA extracted from the liver tissue of rat was served as template, then used the ds exon7 of the p53 gene as template to prepare ss probes by use of single-primer PCR and asymmetric PCR.Then we used the Dig-labeled dNTP instead normal dNTP to synthesize non-radioactive ss probes by PCR method employed the purified exon7 of the p53 gene as template. The probes labeled by PCR could be seen after the agarose gel electrophoresis, but its speed was slower than normal because of the affiliation of Dig molecular, this illuminated the success of labeling.§2. Identifying the effect hybridized by ss probesIn this part , rats were administered with benzidine by ip. injection and genomic DNA of lung were extracted, after RDPCR and transfer, hybridized with ss probes and ds probes, then identifiedthose two results, we found the sensibility and the specificity of single-strand probes (ss probes) were better than ds probes, the aim gene strips were clear and the background almost can't seen, so we thought ss probes were more suit for RDPCR, applying ss probes would be helpful for expand and develop of RDPCR.Part I I AppI i cat ion of ss probes RDPCR technique to detect DNAdamage of rats p53 geneIn this part, Rats were administered with benzidine, potassium dichromate and organic extracts of raw water, genomic DNA of several organs were extracted and amplified using RDPCR, and hybridized with the ss probes.Then we found positive result in liver, lung and bladder organization administered with benzidine, liver and lung organization administered with potassium dichromate, liver and kidney organization administered with organic extracts of raw water, those results is propitious to clarify the molecular mechanism of carcinogensis of those environmental carcinogens, and confirmed again that the ss probes RDPCR in vivo is reasonable and feasible.