制备异源双链增强型绿色荧光蛋白质粒，用于体内错配修复活性测定。
Preparation of heteroduplex enhanced green fluorescent protein plasmid for in vivo mismatch repair activity assay.
机构信息
Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, 325035 Wenzhou, People's Republic of China.
出版信息
Anal Biochem. 2009 May 1;388(1):167-9. doi: 10.1016/j.ab.2009.02.020. Epub 2009 Feb 25.
Abstract
Preparation of heteroduplexes in large quantities with high purity is essential for the measurement of DNA mismatch repair (MMR) activity. Here we report a rapid, less labor-intensive method for the preparation of a heteroduplex plasmid that expresses the enhanced green fluorescent protein (EGFP) if the mismatch is repaired correctly. The method involves the use of a wild-type and a mutated EGFP expression plasmid and a few steps of enzymatic digestion. When the constructed heteroduplex EGFP plasmid was transfected into MMR-proficient and -deficient cell lines, the number of EGFP-expressing cells was much higher in the MMR-proficient cells than in the MMR-deficient cells, suggesting that the heteroduplex can be used for MMR activity assay in live model systems.
摘要
大量制备具有高纯度的异源双链体对于测量 DNA 错配修复 (MMR) 活性至关重要。在这里，我们报告了一种快速、劳动强度较小的方法，用于制备异源双链体质粒，如果错配得到正确修复，该质粒会表达增强型绿色荧光蛋白 (EGFP)。该方法涉及使用野生型和突变型 EGFP 表达质粒以及几个酶切步骤。当构建的异源双链体 EGFP 质粒转染到 MMR 功能正常和功能缺失的细胞系中时，在 MMR 功能正常的细胞中表达 EGFP 的细胞数量远高于 MMR 功能缺失的细胞，这表明异源双链体可用于活细胞模型系统中的 MMR 活性测定。
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