Shimpi Gaurav G, Vargas Sergio, Wörheide Gert
Department of Earth and Environmental Sciences, Palaeontology & Geobiology, Ludwig-Maximilians-Universität München, Richard-Wagner-Str. 10, 80333, Munich, Germany.
Department of Earth and Environmental Sciences, Palaeontology & Geobiology, Ludwig-Maximilians-Universität München, Richard-Wagner-Str. 10, 80333, Munich, Germany; GeoBio-Center(LMU), Richard-Wagner-Str. 10, 80333, Munich, Germany; SNSB -Bavarian State Collections of Palaeontology and Geology, Richard-Wagner-Str. 10, 80333, Munich, Germany.
Anal Biochem. 2018 Sep 1;556:35-39. doi: 10.1016/j.ab.2018.06.020. Epub 2018 Jun 22.
We present efficient and reproducible parallel strategies for preparing large quantities of pure heteroduplex plasmids containing defined mismatches. The strategies described involve the use of synthetic oligonucleotides, the commercially available pGEM-T plasmid, and nicking enzymes to prepare prerequisite ssDNA. Alternatively, bacterial packaging cell lines containing an engineered phagemid construct to produce ssDNA without the need of a helper phage were utilized, hence providing added flexibility and choice. These integrated approaches help to construct different mismatch substrates of choice in large quantities, thus enhancing the usability of mismatch repair assays and extending their range and accessibility to wider research groups.
我们提出了高效且可重复的平行策略，用于制备大量含有特定错配的纯异源双链质粒。所描述的策略包括使用合成寡核苷酸、市售的pGEM-T质粒和切口酶来制备所需的单链DNA。另外，还利用了含有工程化噬菌粒构建体的细菌包装细胞系来产生单链DNA，而无需辅助噬菌体，从而提供了更大的灵活性和选择。这些综合方法有助于大量构建不同的所需错配底物，从而提高错配修复检测的可用性，并将其范围和可及性扩展到更广泛的研究群体。
