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

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。

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

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.