单链构象多态性分析检测脂蛋白脂酶基因变异  

Analysing and Detecting the Gene Mutation of Lipoprotein Lipase by the Method of PCR-SSCP.

作　　者：倪培华[1] 吴洁敏[1] 项佑贵[2] 陈铭生[2] 

机构地区：[1]上海第二医科大学附属瑞金医院医学检验系,200025 [2]上海第二医科大学附属仁济医院检验科,200001

出　　处：《诊断学理论与实践》2002年第2期84-87,共4页Journal of Diagnostics Concepts & Practice

摘　　要：目的:建立聚合酶链反应-单链构象多态性（PCR-SSCP）技术检测脂蛋白脂酶（LPL）基因Hind　Ⅲ多态性、Ser447Ter多态性及-93T/G基因突变的方法。方法:5μl Hind Ⅲ多态性或-93T/G基因突变的PCR产物和5μl甲酰胺变性液混匀变性,室温下用8％的聚丙烯酰胺凝胶电泳后银染。5μl　Ser447Ter多态性的PCR产物和2μl甲酰胺变性液混匀变性,25℃左右用8％的聚丙烯酷胺凝胶电泳后银染。结果:PCR-SSCP成功检出LPL的Hind Ⅲ多态性、Ser447Ter多态性及-93T/G基因突变,并检出第9内含子50位一个G碱基缺失和启动子区域-85位一个C-T碱基置换即-85C/T。同时PCR-RFLP验证了200例标本的PCR-SSCP检测结果,两种方法检测已知基因变异时的结果完全一致。结论:PCR-SSCP法既能检测已知的LPL基因变异,也可检出未知的LPL基因变异,是临床实验室开展基因诊断的一种简便、有效的方法。Objective: To establish a method of PCR-SSCP to detect Hind Ⅲ polymorphism, Ser447Ter polymorphism, -93T/G gene mutation of lipoprotein lipase gene. Methods; 5μl PCR production of Hind Ⅲ polymorphism or-93T/G gene mutation was mixed with 5 μl denatured formamide solution. Then the denatured mixture was electrophoresed in 8% polyacrylamide gel at room temperature. 5 μl PCR production of Ser447Ter polymorphism was mixed with 2 μl denatured formamide solution. The denatured mixture was electrophoresed in 8% polyacrylamide gel at 25℃. Then the polyacrylamide gel was stained by AgNO3. Results: Lipoprotein lipase gene Hind Ⅲ polymorphism, Ser447Ter polymorphism and-93T/G gene mutation were successfully detected by PCR-SSCP. A single base C-T transition at nucleotide-85 of the LPL gene promoter and a single base G missing mutation (GGG-GG) at nucleotide 50 of intron 9 were also found. At the same time, PCR-RFLP assay was used to verify the result of LPL genotype detected by means of PCR-SSCP assay in 200 individuals. No significant difference was found between the results of LPL genotype analyzed using two methods. Conclusions: PCR-SSCP could be used to detect the known mutations and polymorphisms of LPL gene as well as the unknown mutations. It is simple and effective to use for clinical molecular diagnostic laboratory.

关 键 词：脂蛋白脂酶 基因突变 基因多态性 单链构象多态性 基因诊断 

分 类 号：R446.1[医药卫生—诊断学]