变性高效液相色谱检测IRS-2基因3′-非翻译区单核苷酸多态性

Detection of single-nucleotide polymorphisms in insulin receptor substrate-2 gene 3′-untranslated region by denaturing high-performance liquid chromatography

目的 :探讨变性高效液相色谱 (DHPLC)在胰岛素受体底物 2 (IRS 2 )基因 3′ 非翻译区单核苷酸多态性 (SNP)检测中的应用。方法 :采用聚合酶链式反应 (PCR) ,DHPLC ,单链构象多态性 (SSCP)和DNA序列分析对 30例正常对照和 30例 2型糖尿病患者IRS 2基因 3′ 非翻译区进行SNP和突变检测。结果 :PCR DHPLC检测出 4例PCR SSCP分析未能发现的 2型糖尿病患者IRS 2基因 3′ 非翻译区的SNP ,并得到测序验证。结论 :DHPLC是一种快速、自动化程度高、操作简单、检测片段长、准确率高的SNP检测技术。它为进一步研究IRS 2基因 3′ 非翻译区的变异与 2型糖尿病的关系奠定了基础。

Objective To explore the use of denaturing high-performance liquid chromatography(DHPLC) in detecting single-nucleotide polymorphisms(SNPs) of insulin receptor substrate-2(IRS-2) gene 3′-untranslated region (3′-UTR). Methods We detected the SNPs and mutation of IRS-2 gene 3′-UTR sequence, with polymerase chain reaction (PCR), DHPLC, single-strand conformation polymorphism (SSCP), and DNA sequence analysis respectively in 30 Type 2 diabetic subjects and 30 healthy controls. Results The SNPs of IRS-2 gene 3′-UTR in 4 patients with Type 2 diabetes mellitus were detected by PCR-DHPLC and were identified by DNA sequencing. Conclusion DHPLC is a rapid and automated technology, which is simpler and more accurate for detecting longer fragment of DNA than SSCP. The present method lays a foundation for further studying the relationship between the mutation of IRS-2 gene 3′-UTR and Type 2 diabetes mellitus.