用外切酶 I 和热不稳定碱性磷酸酶处理 PCR 产物可提高联合亚硫酸氢盐限制分析的可视性。

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.

机构信息

Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

出版信息

Biochem Biophys Res Commun. 2010 Aug 27;399(3):422-4. doi: 10.1016/j.bbrc.2010.07.093. Epub 2010 Jul 30.

DOI:10.1016/j.bbrc.2010.07.093

PMID:20674551

Abstract

DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

摘要

DNA 甲基化在基因表达调控中起着至关重要的作用。异常启动子过度甲基化是人类癌症中肿瘤抑制基因失活的重要机制。联合亚硫酸氢盐限制分析（COBRA）是一种广泛用于鉴定特定 CpG 位点 DNA 甲基化的方法。在这里，我们报告说，核酸外切酶 I 和热不稳定碱性磷酸酶可用于 COBRA 的 PCR 纯化，在限制性消化后提高凝胶电泳的可视性。在用 BstUI 和 TaqI 分别在 60°C 或 65°C 的高温下进行限制性消化时，可以观察到这种改进。这种简单的方法可以替代使用 spin columns 或酚/氯仿提取进行 DNA 纯化。它还可以应用于其他情况，例如当 PCR 产物被嗜热衍生的限制酶消化时，例如 PCR 限制片段长度多态性（RFLP）分析。