DNA甲基化敏感位点(CCGG)文库克隆载体的构建

Construction of Library Cloning Vector with DNA Methylation Sensitive Site( CCGG)

目的:旨在建立一种简便易行的DNA甲基化文库构建载体,用于筛选细胞中具有甲基化敏感性CCGG位点的DNA片段。方法:根据HpaⅡ/MspⅠ酶切体系对甲基化酶敏感性不同的特点,通过在PCR引物中添加CCGG酶切位点的方法,将扩增得到的目的片段连接到无CpG甲基化的质粒载体上。结果:成功构建了含双CCGG位点的pCpGfree-HpaⅡ/MspⅠ-1质粒载体。结论:所构建的pCpGfree-HpaⅡ/MspⅠ-1质粒载体CCGG位点未发生甲基化,故可以用于构建含有甲基化CCGG序列的DNA甲基化文库。

Objective：The purpose of this study is to establish a simple vector for DNA methylation library building for the screening of DNA fragments that have methylation sensitive site （CCGG）. Method： Based on the sensitive differences of the restriction enzyme pair Hpa Ⅱ/Msp I on the CCGG, the CCGG site was incorporated into the PCR primers and the amplified PCR fragments were ligated into a vector that lacks the CpG islands. Result： pCpGfree - HpaⅡ/Msp I - 1 vector with two CCGG sites was successfully created. Condusion： The CpG islands free vector pCpGfree - HpaⅡ/Msp I - 1 was successfully constructed and evaluated for screening of DNA methylation status, thus, it can be used for DNA methylation sreening.