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研究胰岛素受体底物 1(IRS 1)基因 5′ 调控序列与 2型糖尿病的关系。采用PCR SSCP技术扫描 76例 2型糖尿病患者和 78例正常对照IRS 1基因 5′ 调控区 ,结合应用PCR 变性聚丙烯酰胺凝胶电泳和银染法 ,检测IRS 1基因 5′ 调控序列CAG富含区的插入 /缺失多态性 ;以检获的正常和变异样品基因组DNA为摸板 ,用高保真pfuDNApolymerase进行扩增 ,采用亚克隆技术 ,将扩增片段分别插入pCATBasic质粒中。通过限制性内切酶酶切并结合变性聚丙烯酰胺凝胶电泳和银染分析后 ,根据迁移率不同筛选含有不同等位基因的重组质粒并进行测序。结果发现人IRS 1基因 5′ 调控区CAG富含区存在以三核苷酸为单位的多种插入 /缺失变异。共检测出 7种基因型和 6种等位基因 (T1～T6 ) ,其中等位基因T5 ,T6仅见于 2型糖尿病组。
To study the relationship between 5’regulatory sequence of insulin receptor substrate 1 (IRS 1) gene and type 2 diabetes. PCR SSCP technique was used to scan the 5 ’regulatory region of IRS 1 gene in 76 patients with type 2 diabetes mellitus and 78 normal controls. Combined with PCR denaturing polyacrylamide gel electrophoresis and silver staining, the 5’ regulatory region of IRS 1 gene CAG was detected The genomic DNAs of the normal and mutant samples were used as the template to amplify with high fidelity pfuDNApolymerase. The subclones were inserted into pCATBasic plasmids respectively. Restriction endonuclease digestion and combined with denaturing polyacrylamide gel electrophoresis and silver staining analysis, according to different mobility screening of different alleles containing recombinant plasmids and sequencing. The results showed that a variety of insertion / deletion variations in trinucleotide units existed in the CAG-rich region of the 5 ’regulatory region of human IRS 1 gene. A total of 7 genotypes and 6 alleles (T1 ~ T6) were detected. The alleles T5 and T6 were only found in type 2 diabetes mellitus.