| As one of the diseases causing severe trouble to human health, malignant tumor has ranked the first one to cause death with its increasing incidence. With the deterioration of our living environment and the heave pressure of living and competing, the tumor incidence has rising year by year. As one of the common malignant tumors, gastric cancer is recognized as the most severe one in enteron tumors. The early diagnosis plays a key role in curing gastric cancer. However, the unobvious symptom and the difficulties lying in the early diagnosis largely affect the effect of the treatment. The discovery of idio-tumor marker will be a great step for the early diagnosis and screening of gastric cancer.SNP, the most ordinary one in human heredity variation, has accounted to more than 90% of the already known polymorphism and become the genetic marker in human genes with the highest density and incidence, thus SNP is regarded as genetic marker of the new generation after the finding of microsatellite and has an important value in researching and application in many fields of Medical Genetics, Pharmacogenetics, Individual Medicine, Genetic Disease and Disease Diagnosis etc.. In recent years, researches have discovered that SPN sites of different genes have close relations with the occurrence and development of cancer. To apply SNP as the bio-marker has been proved to have high feasibility in the early cancer detecting.The current methods in researching SNP are mainly conformation polymorphism, denaturing gradient gel electrophoresis, RFLP (restriction fragment length polymorphism) and TaqMan probe method. However, they can not be used in automatic screening of large scale for their disadvantages of low flux, disability to realize automation and high detecting cost. MassARRAY system is a multi-functional system applying mass spectrometric detection technology to analyze nucleic acid and perfectly combining the high sensitivity and accuracy of PCR, which is in conformance with the rapid development trend of medical researches of disease genes and mechanism with advantages of flexible choice of sites, correct detecting results, high flux and low cost. This essay will apply the relation of detecting gene SNP and gastric cancer to detect the SNP frequency of paraffin embedding tissue, discuss the relations of SNP and gastric cancer and evaluate the possibility of SNP as a marker of the early diagnosis and screening tumor.This essay chooses 27 sites from 10 genes of APC、KRAS、PIK3CA、PTEN、CTNNB1、EGFR、TP53、AKT、ERBB2、and PSCA, extracts DNA of genome from gastric cancer paraffin embedding tissue, uses the primer made by Assay Designer software and the probe to amplify the target gene fragment and detects by using MassARRAY molecular array analysis system so as to discover the high frequency of variation of 5 genes APC、KRAS、PIK3CA、PSCA and PTEN and 8 sites.In order to continue the task that MassARRAY system can not finish, realize the method of detecting different variation sites in one time, and lay the foundation for diagnosis research, the experiment applies PCR primer designed by PYRO ASSAY DESIGN to amplify the target area of PIK3CA, PSCA and PTEN, and detect the sites by pyrosequencing and Sanger sequencing to further prove MassARRAY system, to find out that the pyrosequencing can realize the one time detecting to PIK3CA sites and realize the detecting of PSCA which is not suitable for PTEN.The research detected and analyzed the 27 variation sites from 10 genes from the paraffin embedding of gastric cancer patients, among which 5 genes APC、KRAS、PIK3CA、PSCA and PTEN and 8 sites have high frequency of mutation and have the potential value to become tumor markers. The detection rate of PTEN is comparatively low because of the influence of primer design and PCR amplification. |
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