Development Of Gene Analysis Metods Based On Capillary Electrophoresis And Its Application In The Hereditary Background Investigation Of Cancer

Cancer is one of the genetic diseases related to heredity. Hereditarybackground of cancer will be helpful to prevent cancer in early stage and genetictherapy on cancer.In the dissertation, a single-strand conformation polymorphism analysis withcapillary electrophoresis for detection of the mutations in mismatch repair geneshas been developed employing homemade linear polyacrylamide as sievingmedium. The effects of temperature and the addition of glycerol on SSCP-CEanalysis were investigated. The results show the effect of temperature depends onthe base sequence of single-strand DNA and the properties of the separated bufferwere changed by the reaction between glycerol and boric acid, which improvedthe resolution of single-strand DNA.PCR based methylation-sensitive restriction enzymes analysis andmethylation-sensitive SSCP analysis for detection of the hMLH1 promotormethylation were set up with laser-induced fluorescence capillary electrophoresis.On the ABI 310 genetic analyzer, a method for the analysis ofmicrosatellite instability and loss of heterozygosity was developed employinga homemade kit to replace the commercial kit and multiplex-fluorescencedetection. The effects of temperature and the addition of urea onmicrosatellite analysis and the length of microsatellite fragments wereinvestigated. The second-structure of microsatellite fragments was eliminatedcompletely by the addition of 8 mol/L urea and at 60℃. As a result, the lengthof microsatellite fragments inclined to their actual length. Based on thismethod, five microsatellite loci were simultaneously detected andone-bp-shift was detectable and loss of heterozygositywas exactly judged.These above-mentioned methods based on capillary electrophoresis wereapplied to the routine genetic analysis of clinical specimens including familialgastric cancer, multiplexcancer and various sporadic cancers. Three mutations ofhMLH1 exon8, exon12 and exon16 were detected. The T1775G mutation ofexon16 is a new mutation type of hMLH1 gene, which was never reported in theworld. Moreover, it could be the hot position of hMLH1 mutation in Chinesefamilial gastric cancer. The relationship between hMLH1 mutation and multiplexcancer is closer than that in single cancer. In the sporadic cancers, the major factorof the inactivation of hMLH1 gene is the hMLH1 promoter methylation; thehMLH1 mutation is minor. In addition, the rate of spontaneous mutation in K-rasoncogene and microsatellite instability increased owing to hMLH1 inactivation.