Study On The Method Of Gene Mutation Detection By Capillary Electrophoresis And Its Application In Lung Cancer

Lung cancer is one of the most common malignant tumor,and its occurrence is closely related to gene mutation.Therefore,it is essential to establish a rapid and sensitive detection method to analyze gene mutation.Currently many methods are used to detect gene mutation. Restriction fragment length polymorphism is often together with PCR to detect gene mutation,and it needs electrophoresis for analysis of gene mutation.ButCapillary electrophoresis is simple and sensitive,and it has been widely used in the field of gene mutation detection.This paper has two parts to investigate the detection of gene mutation by capillary electroph0resis in our experiment.In the first part,firstly we systematically investigated the separation ability of sieving matrixe PEO to separate different size and range DNA fragments with vacuous and covalent coating capillary column.The effects of concentration of polyethyleneoxide,running pH, running temperature and running voltage on dsDNA analysis were investigated,pUC19 DNA/MspⅠ(HpaⅡ) DNA Marker separation of PEO under vacuous capillary column were not well.EO can separate pUC19 DNA/MspⅠ(HpaⅡ) DNA Marker pretty well under coating,capillary column.The system we have set up under covalent coating capillary column can simultaneously separate dsDNA(30bp-600bp) and it is suitable for gene mutation analysis of RFLP.Use the pUC19 DNA/MspⅠ(HpaⅡ) Marker standard DNA fragments as study target. Compared to the molecular weight 300000 of PEO,the separation efficiency of the molecular weight 4.5 million of PEO was poor.So the optimum separation condition was applied in analyzing RFLP of PCR products of codon 248 and 249 in p53 and codons 12 in H-ras and K-ras gene.The optimum separation condition was 3.0%PEO(Mr=300 000),running pH at 8.2,running voltage at 15 kV,running temperature at 15℃.We detect the mutation rates of p53 and ras gene in different lung cancer tissues and discuss their effects and positions in the pathogenesis of lung cancer.The pathological type of lung cancer are non small cell lung cancer and small cell lung cancer.There is no gene mutation in 38 normal tissues of 76 samples,the mutation rates of p53 gene in NSCLC and SCLC are 75.86%and 77.78% separately,and the mutation rates of ras gene in NSCLC and SCLC are 51.72%and 0 separately.And the experiment was completed in about 10 minutes.In the second part,the optimum separation condition was applied in analyzing RFLP of PCR product of codon 249 in p53 gene.The optimum separation condition was 2.0%MC, running pH at 8.0,running voltage at 7.5kV,running temperature at 25℃.Detect the 249 coden of lung cancer and normal lung tissue p53 Gene point Mutation by PAGE and CE simultaneously.The detected mutant ratios by PFLP-CE and RFLP-PAGE are 31.03%and 20.69%separately in non small cell lung cancer.The detected gene mutant ratio by RFLP-CE is obviously higher than RFLP-PAGE.There is no one gene mutation of non small cell lung cancer and normal lung tissues,and in non small cell lung cancer the codon 249 in p53 gene mutation has nothing to do with different degree of lung cancer.And the experiment was completed in about 20 minutes.The established CE method is suitable to separate DNA fragments which is smaller than 600 bp.The detection time of CE which is used in gene mutation detection is short,and the detection rate is high.A fast,high resolving power method was initially established in clinical diagnosis of lung cancer.