Establishing The Method To Detect The DNA Damage Of Ras Gene AND Studying The Mechanism Of Carcinogenesis Of Environmental Carcinogen

DNA damage is a detectable key step during the early stage of environmental carcinogenesis and its detection has very important toxicological significance. DNA damage could be regarded as an important biomarker of exposure and effect to evaluate the genotoxicity of environmental chemicals and risk assessment.DNA damage occurr non-randomly, and different genes, or even different base site of the same gene would be different in susceptivity and repair rate to the damage, So it would be significant to detect the DNA damage of a given gene at single-nucleotide resolution with a certain method.There are lots of conventional methods to detect DNA damage, but they can't do that at single-nucleotide resolution at all. Since some new methods have coming, we can resort a new method named LMPCR, which could detect the DNA damage of a certain gene at single-nucleotide resolution.The randomized terminal linker-Dependent PCR (RDPCR), is a novel technique which have been designed by us on the basis of LMPCR. It could detect any kinds of damage which could stop the extension of polymerase theoretically. We have established the new method to detect the DNA damage of p53 gene.As a representative of proto-oncogenes, ras gene plays animportant role in carcinogenesis and cell differentiation. It will be helpful in tumor prevention, etiology and other aspects to study ras gene. Furthermore, it is reported that the DNA damage of ras gene is correlate with the mutagenesis/carcinogenesis of environmental chemicals. As we have known, ras gene family is a totally different gene with/755 in the structure and function, so we have to do lots of work to develop the method.In our study, we have prepared the single-stranded probes of ras gene and established the method using RDPCR to detect the DNA damage of ras gene successfully. In addition, we have optimized the technique in some aspects. Furthermore, we have detected the DNA damage of ras gene induced by environmental carcinogens and evaluate the molecular mechanism of their carcinogenesis.Part â… Preparation of single-stranded probes of ras geneIn this part, we study the exon 1 of N-ras gene firstly. With the genome DNA as template, and directed by primers designed by ourselves, the 138bp segment (double strand) of the exon1 of the N-ras gene was successfully amplified, then it is used as template to prepare the Dig-labeled non-radioactive ss probes using single-primer PCR and asymmetric PCR respectively.Following this way, we have prepared ss probes of exon 2 of N-ras gene, exon 1 and exon 2 of H-ras gene successfully.Part â…¡ Establishment of the RDPCR method to detect theDNA damage of ras geneAs no reports have been shown on the Establishment of the RDPCR method to detect the DNA damage of ras gene, in this part, the genomic DNA was digested completely by restriction endonuclease Ddel, then repeatedly extended by the primer PI, ligated by a common double-strand linker which has a randomized 3' overhang, and amplified by the primer of linker (PL) and P2. The PCR products were electrophoresed on agarose gel, vacuum transferred to positive charged nylon membranes, and hybridized with the Dig-labeled ss probe of exon 1 of N-ras gene. Furthermore, we have optimized the RDPCR technique during this way.The clear bands can be seen in expected migration positions, which means that we have succeeded in developing the RDPCR to detect the DNA damage of exon 1 of N-ras gene.On the basis of these results, we also develop the RDPCR to detect the DNA damage of exon 2 of N-ras gene, exon 1 and exon 2 of H-ras gene.Part â…¢The detection of DNA damage of ras gene induced by environmental carcinogens and evaluation of themolecular mechanism of their carcinogenesisIn this part, TK6 cell was treated with some environmental carcinogens and organic extracts of chlorinated drinking water.genomic DNA is extracted and amplified using RDPCR, and hybridized with the ss probes of different ras genes. The results show that DNA damage of different ras genes can be detected.It indicates that Potassium dichromate can cause the DNA damage of promoter and intron 1 of N-ras gene, which should be the key point of its carcinogenesis mechanisms. In addition, the result could reflect the merit of RDPCR to detect the DNA damage of complete sequences of a given gene.We find a positive result induced by treated chlorinated drinking water, but a negative result by raw water. It indicates that DBP should have mutagenesis. And this result embodies the characteristic of RDPCR to detect the DNA damage at single-nucleotide resolution in specific gene induced by complex unknown environmental sample.In conclusion, we have found some new positive result induced by DCA, TCA, Benzidine, Benzo(a)pyrene, etc. The appearance that different carcinogens can induce DNA damage of different ras genes indicates that ras gene would be a good biomarker of exposure and effect of environmental carcinogens.The similar results to our study have not been reported till now. The results should be significant in further studying of the molecular mechanism of these environmental carcinogens.