Laboratoire Jean Perrin, UMR 8237, CNRS, Sorbonne Universités, UPMC Université Paris 06, Paris, France.
Micalis Institute, Institut National de la Recherche Agronomique, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.
Nat Protoc. 2019 Nov;14(11):3126-3143. doi: 10.1038/s41596-019-0215-x. Epub 2019 Sep 25.
Mutations are the driving force of evolution and the source of important pathologies. The characterization of the dynamics and effects of mutations on fitness is therefore central to our understanding of evolution and to human health. This protocol describes how to implement two methods that we recently developed: mutation visualization (MV) and microfluidic mutation accumulation (µMA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (µMA) to be followed directly in individual cells of Escherichia coli. MV provides a quantitative characterization of the dynamics of mutation occurrences, and µMA allows precise estimation of the distribution of fitness effects (DFEs) of mutations. Both methods use microfluidics and time-lapse microscopy, and a fluorescent mismatch repair (MMR) MutL protein is used as a marker for nascent mutations. Here, we present a single protocol describing how to implement the MV and µMA methods, including detailed procedures for microfluidic setup installation, data acquisition and data analysis and interpretation. Using this procedure, the microfluidic setup installation can be completed within 1 d, and automated data acquisition takes 2-4 d.
突变是进化的驱动力，也是重要病理学的源头。因此，对突变对适应性的动态和影响进行特征描述，是我们理解进化和人类健康的核心。本方案描述了如何实施我们最近开发的两种方法：突变可视化（MV）和微流控突变积累（µMA），这两种方法可直接在大肠杆菌的单个细胞中跟踪由 DNA 复制错误引起的突变（MV）的发生和 MA 期间细胞适应性的演变（µMA）。MV 提供了突变发生动态的定量特征描述，而 µMA 则允许对突变适应性影响分布（DFE）进行精确估计。这两种方法都使用微流控和延时显微镜，荧光错配修复（MMR）MutL 蛋白被用作新生突变的标记。在这里，我们提供了一个描述如何实施 MV 和 µMA 方法的单一方案，包括微流控设置安装、数据采集和数据分析与解释的详细步骤。使用此程序，微流控设置安装可在 1 天内完成，自动数据采集需要 2-4 天。
