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Updated: Nov 1, 2025
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis
Ondrej Belan1, George Moore2,3, Artur Kaczmarczyk2,3
1DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
None:
Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).
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