Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

Ondrej Belan; George Moore; Artur Kaczmarczyk; Matthew D. Newton; Roopesh Anand; Simon J. Boulton; David S. Rueda

STAR Protocols (Jun 2021)

Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

Ondrej Belan,
George Moore,
Artur Kaczmarczyk,
Matthew D. Newton,
Roopesh Anand,
Simon J. Boulton,
David S. Rueda

Affiliations

Ondrej Belan: DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Corresponding author
George Moore: Department of Infectious Disease, Faculty of Medicine, Imperial College London, London W12 0NN, UK; Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London W12 0NN, UK
Artur Kaczmarczyk: Department of Infectious Disease, Faculty of Medicine, Imperial College London, London W12 0NN, UK; Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London W12 0NN, UK
Matthew D. Newton: Department of Infectious Disease, Faculty of Medicine, Imperial College London, London W12 0NN, UK; Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London W12 0NN, UK
Roopesh Anand: DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Simon J. Boulton: DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Corresponding author
David S. Rueda: Department of Infectious Disease, Faculty of Medicine, Imperial College London, London W12 0NN, UK; Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London W12 0NN, UK; Corresponding author

Journal volume & issue: Vol. 2, no. 2
p. 100588

Abstract

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Summary: 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).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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