胰岛素受体底物—1基因5’—调控序列CAG富含区域基缺失／插入的研究

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为模板，用高保真pfu DNA polymerase进行扩增，采用亚克隆技术，将扩增片段分别插入pCAT Basic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。

Study on base insertion/deletion of CAG-rich region in insulin receptor substrate-1 5'-regulatory sequence]

To study the relationship of the polymorphism of the insulin receptor substrate-1 (IRS-1) gene 5'-flanking regulatory sequence and Type 2 diabetes, the IRS-1 gene 5'-flanking regulatory sequence was scanned by PCR-SSCP in 78 healthy control subjects and 76 Type 2 diabetic subjects. Applying PCR-denatured polyacrylamide gel electrophoresis and silver staining, the insertion/deletion polymorphism of the CAG-rich region was analyzed. The genome DNA of the normal and variant subjects was amplified with high-fidelity pfu DNA polymerase. The purified and digested target fragments were then subcloned into the pCAT Basic vector. Each allele was identified according to the mobility by the restrictive endonuclease digestion of the recombinant combined with denatured polyacrylamide gel electrophoresis and silver staining, and finally the constructive plasmids containing different alleles were analyzed by DNA sequencing. Firstly, we found several insertion/deletion variations in the CAG-rich region of IRS-1 gene. Secondly, 7 genotypes and 6 alleles(T1-T6) in this site were detected. Moreover, T5 and T6 were only observed in Type 2 diabetic group.