Study on base insertion/deletion of CAG-rich region in insulin receptor substrate-1 5′-regulatory sequence / 中南大学学报(医学版)

ABSTRACT

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.