Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
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DOI：
10.3791/61236
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发表时间：
2020-05-01
影响因子：
1.2
通讯作者：
Price, Allen C.
中科院分区：
文献类型：
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作者：
Matozel, Emily K.;Dale, Nathaniel;Price, Allen C.
定点DNA切割(SSDC)是许多细胞过程中的关键步骤，也是基因编辑的关键。这项工作描述了一种能够同时测量多个单DNA分子中SSDC的动力学分析方法。在微流控流动通道中制备微珠拴系底物DNA，每个DNA包含靶序列的单个拷贝。外部磁铁对顺磁珠施加微弱的力。通过使用广视场、低倍率物镜在暗场成像下可视化微珠，可以监测多达1,000个个体DNA的完整性。注射限制性内切酶NdeI启动切割反应。视频显微镜被用来记录每一次DNA切割的准确时刻，方法是观察相关的珠子在其中向上移动和离开物镜焦面的帧。逐帧珠数量化反应，指数拟合度决定反应速率。这种方法允许在单个实验中收集关于单分子SSDC反应的定量和统计上有意义的数据。
Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.