Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, 325035 Wenzhou, People's Republic of China.
Anal Biochem. 2009 May 1;388(1):167-9. doi: 10.1016/j.ab.2009.02.020. Epub 2009 Feb 25.
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.
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。
