基于毛细管电泳的基因分析方法及肿瘤遗传背景研究
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摘要
恶性肿瘤是与遗传明确相关的“基因病”之一,肿瘤遗传背景研究对肿瘤发生的早期预防及肿瘤基因治疗具有重要意义。
本论文以自制线性聚丙烯酰胺为分离介质,建立了检测错配修复基因突变的单链构象多态性-毛细管电泳分析方法,并系统研究了温度、甘油添加剂等因素对单链DNA 构象分析的影响。结果表明:温度对不同碱基序列的单链DNA 构象影响不同;甘油通过与分离介质中的硼酸反应,改变了分离介质的特性,从而提高了单链DNA构象的分离。
在毛细管电泳上建立了检测hMLH1 基因启动子区甲基化的限制性内切酶PCR 法和甲基化敏感的单链构象分析法,提高了甲基化的检出率。
在ABI 310 型基因分析仪上,用多色荧光标记检测和自制分析试剂盒,建立了检测微卫星不稳定性和杂合性缺失的方法,并系统研究了尿素变性剂和温度对微卫星分析和微卫星片段长度的影响。结果表明在60℃下,分离介质中添加8mol/L尿素可以完全消除二级结构,使微卫星片段的标定长度趋于其真实长度;建立的方法可以同时检测5 个微卫星位点,而且能检测出单碱基漂移和定量检测杂合性缺失。
利用上述基于毛细管电泳的基因分析平台,筛查了家族性胃癌、多原发癌和散发性癌症中错配修复基因hMLH1、hMSH2 和癌基因K-ras 的突变,hMLH1 基因启动子甲基化以及微卫星不稳定性情况。结果在hMLH1 基因的exon8、exon12 和exon16 上发现突变,其中exon16 T1775G点突变是国际上未曾报道过的一种新的hMLH1 基因突变形式,而且可能是我国胃癌家系突变热点。此外还发现hMLH1 基因突变与多原发癌的发生关系密切;散发性肿瘤中,造成错配修复基因hMLH1 功能失活的主要原因是hMLH1 启动子甲基化,hMLH1 基因突变只起次要作用;而且有错配修复基因hMLH1 功能缺陷容易导致微卫星不稳定发生和癌基因K-ras 突变。
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 were
applied 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.
本论文以自制线性聚丙烯酰胺为分离介质,建立了检测错配修复基因突变的单链构象多态性-毛细管电泳分析方法,并系统研究了温度、甘油添加剂等因素对单链DNA 构象分析的影响。结果表明:温度对不同碱基序列的单链DNA 构象影响不同;甘油通过与分离介质中的硼酸反应,改变了分离介质的特性,从而提高了单链DNA构象的分离。
在毛细管电泳上建立了检测hMLH1 基因启动子区甲基化的限制性内切酶PCR 法和甲基化敏感的单链构象分析法,提高了甲基化的检出率。
在ABI 310 型基因分析仪上,用多色荧光标记检测和自制分析试剂盒,建立了检测微卫星不稳定性和杂合性缺失的方法,并系统研究了尿素变性剂和温度对微卫星分析和微卫星片段长度的影响。结果表明在60℃下,分离介质中添加8mol/L尿素可以完全消除二级结构,使微卫星片段的标定长度趋于其真实长度;建立的方法可以同时检测5 个微卫星位点,而且能检测出单碱基漂移和定量检测杂合性缺失。
利用上述基于毛细管电泳的基因分析平台,筛查了家族性胃癌、多原发癌和散发性癌症中错配修复基因hMLH1、hMSH2 和癌基因K-ras 的突变,hMLH1 基因启动子甲基化以及微卫星不稳定性情况。结果在hMLH1 基因的exon8、exon12 和exon16 上发现突变,其中exon16 T1775G点突变是国际上未曾报道过的一种新的hMLH1 基因突变形式,而且可能是我国胃癌家系突变热点。此外还发现hMLH1 基因突变与多原发癌的发生关系密切;散发性肿瘤中,造成错配修复基因hMLH1 功能失活的主要原因是hMLH1 启动子甲基化,hMLH1 基因突变只起次要作用;而且有错配修复基因hMLH1 功能缺陷容易导致微卫星不稳定发生和癌基因K-ras 突变。
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 were
applied 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.
引文
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