Study On The Method Of Gene Mutation Detection By Non Gel Sieving Capillary Electrophoresis And Its Application In Gastric Cancer

Gene mutation is correlated to the pathogenesis of gastric cancer and its development. We should search for a new method to detect the gene mutation, which is a focal point to most experts and also is a crossing science. At present, the most gene mutation method is based on PCR technology. Restriction fragment length polymorphism which is characterized by simplicity, high veracity is often together with PCR to detect gene mutation, and it need electrophoresis for analysis of gene mutation. Slab-gel electrophoresis is characterized by low sensitivity, low automation and long time consuming. It can not be cosmically applied in clinical diagnosis. With the widespread application in the molecular biology of RFLP ether with CE, it becomes high sensitivity, high resolution and high efficiency.We should set up high efficiency method of gene mutation detection, which can provide scientific foundation of technology and method for clinical diagnosis and prewaming of gastric cancer and this is our purpose in this study. We have set up a system suitable for RFLP-CE and applied to the gene mutation detection of gastric cancer. We systematically studied the separation ability of different sieving matrixes to separate different size and range DNA standard fragments.This dissertation has three parts. First part is the study background of capillary electrophoresis in gene mutation/polymorphism. That different sieving matrixes separate different DNA fragments in non-gel sieving capillary electrophoresis is studied in second part. The third part is about detection of point mutation in gastric cancer tissues with the suitable capillary electrophoresis system together with RFLP, and we discuss the effect and position of p53 and ras gene in the pathogenesis of gastric cancer.The first part reviewed p53 and ras gene, which were relative to the pathogenesis of gastric cancer and the development of abnormal gene detection. CE technology development mainly concentrated to sieving mechanism, sieving matrixes, column coating and other effects, and the application of gene mutation/ polymorphism with CE.In second part, the separation method of different DNA fragments using different sieving matrixes were investigated by NGS-CE. In order to set up a system of double-strands DNA separation, we systematically investigated the separation ability of different sieving matrixes separating different size and range DNA fragments. First, methyl cellulose was used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler^TM DNA Ladder with capillary column coating. The effects of concentration of methyl cellulose, running pH, running temperature and running voltage on dsDNA(< 600bp)analysis were investigated. Methyl cellulose is an excellent medium and it can separate different size and range DNA fragments; PVP and PEO, which are characterized by dynamic coating, relative smaller molecular weight were used to separate pUC19 DNA/Msp I (Hpa II) DNA Marker, PBR322/BsuRI DNA Marker and FastRuler^TM DNA Ladder in vacuous capillary column. pUC19 DNA/Msp I (Hpa II) DNA Marker and PBR322/BsuRI DNA Marker separation of PVP were not well, but FastRuler^TM DNA Ladder separation of PVP obtained high resolution. PEO can separate pUC19 DNA/Msp I (Hpa II) DNA Marker and FastRuler^TM DNA Ladder pretty well. The system we have set up can simultaneously separate dsDNA (10bp-600bp) and it is suitable for gene mutation analysis of RFLP.In third part, the mutations of codon 175 and 245 in p53 and codons 12 in H-ras and K-ras were simultaneously detected by NGS-CE-RFLP. Genomes of gastric cancer patients were extracted. The extracted genomes DNA had better concentration and purification. The purpose DNA fragment were amplified by PCR. PCR product was purified by UNIQ-10 Column DNA Purification Kit to remove salts and primer-dimer. PCR products were cleaved by correct restrictive enzymes. The optimum separation condition was applied in analyzing RFLP of PCR product of p53 and ras gene. The optimum separation condition was 2.0% MC, running pH at 8.0, running voltage at 7.5 kV, running temperature at 25°C. Mutation detection was completed in about 20min.A fast, high resolving power method was initially established in clinical diagnosis of gastric cancer. We detected the mutation rates of p53 and ras gene in different gastric cancer tissue and disguss their effect and position in the pathogenesis of gastric cancer. p53 gene mutation was detected in 8 out of 39 gastric cancer cases (8/39, 20.51%), ras gene mutation was detected in 5 out of 45 gastric cases (5/45, 11.11%). 5 cases(5/39,12.82%) of mutation of p53 175 codon, 3 cases (3/39, 7.69 %)of mutation of p53 245 codon, 1 cases(l/45, 2.22%) of mutation of H-ras 12 codon and 4 cases(4/45, 8.89%)of mutation of K-ras 12 codon were detected. p53 and ras gene mutation were simultaneously detected in 2. The mutation of p53 has no obvious relationship to the pathogenesis areas of gastric cancer, ras gene mutation is mainly in gastric body and cardia. p53 gene mutation plays an important role in some of the gastric cancer patients, ras gene mutation plays no obvious role in the pathogenesis of gastric cancer.