Study On The Methodology Of Gene Mutation Detection And Application In Early Clinical Diagnosis Of Gastric Cancer By Capillary Electrophoresis

The body always is affected by all kinds of carcinogenic factors that lead to tumor development. Tumor is a neoplasm which forms under some DNA impairment in definite chromosome that causes somatic cell mutation and clonally abnormality hyperplasia. Gastric cancer as a malignant tumor of digestive tract with high incidence in our country, its incidence and fatality rate are higher among various malignancies. However, the traditional diagnostic methods of gastric cancer failed to break the bottleneck of early diagnosis that is warning. The post-surgery survival rate of patient decreased for missing the best treatment time. In recent years, with the development of molecular genetics and molecular biology and unceasing advancement of gene engineer technology, the gene study of gastric cancer also got large progression.The first chapter introduces a variety of factors that lead to gastric cancer and the related genes of gastric cancer. In addition, the current gene mutation detection technology and the various factors and parameters effect on capillary electrophoresis (CE) separation are summarized. In this study, related gene mutation of gastric cancer tissue samples was firstly detected by CE combined with single strand conformation Polymorphism assay (SSCP) and restriction fragment length polymorphism (RFLP). Then, peripheral blood of gastric cancer was chosen as detected target which was easily obtained and carried great quantity of genetic information. An early diagnostic method of gastric cancer was established by detecting mutation gene with high incidence to help clinical early diagnosis and guide clinical molecule drug therapy.In the second chapter, target gene fragment of proto-oncogene C-myc, tumor suppressor gene APC and p16were amplified by polymerase chain reaction (PCR). Gene mutation of amplified samples was detected by CE-SSCP, CE-RFLP, polyacrylamide gel electrophoresis (PAGE)-SSCP and sequencing. Then select the mutation genes with high incidence by comparing with the results of CE and traditional methods. Combined with previous experimental data, the statistical results showed that the total mutation rate from high to low:p53(33.3%)> APC (30.0%)>C-myc (20.0%)> K-ras (13.3%)>p16(5.0%). CE-SSCP can be used in detecting gene mutation of clinical samples at large scale. The mutations of p53and APC genes were close to the occurrence and development of gastric cancer.The third chapter and fourth chapter, a fast detection method for analysis of tumor gene mutation in blood samples was established through optimizing the parameter of CE. In contrast to health people, this method was used for detecting gene mutation in peripheral vein blood of patients with gastric cancer. First of all, the genomic DNA was isolated from peripheral blood as template and then four genes with high mutation rate which were choose from experiments of gastric cancer tissue were amplified. Then, the gene mutations of the amplified samples, denatured samples and disgusted samples were detected by direct sequencing analysis, CE-SSCP and CE-RFLP. Then, the total mutation rate was analyzed. The results showed that the total mutation rate was p53(30.1%)> APC (24.6%)> C-myc (19.2%)> K-ras (11.0%). The cumulative detection rate of four genes was84.9%. The detection rate of four genes was improved by simultaneous analysis. Combined with the clinical data, the relationship of gene mutations and case characteristics was analyzed. The statistical results showed that p53and APC gene mutations in gastric cancer were related to its differentiation level. The gene mutation rate of C-myc and K-ras in lymph node metastasis was higher than those without lymph node metastasis group. Four genes involved in gastric carcinogenesis and development processes can be used as an early diagnostic marker of gastric cancer.CE has the advantages of high efficiency, high sensitivity, rapid and accurate in DNA separation and analysis. The development of gastric cancer and gene mutations are closely related. It is evidence-based that establishing clinical early diagnostic method of gastric cancer through detecting gene mutation by CE. The final results of this research showed that the occurrence of gastric cancer could be predicted by detecting the four genes mutations in peripheral venous blood of patients with gastric cancer.