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

STAR Protoc. 2021 Jun 11;2(2):100588. doi: 10.1016/j.xpro.2021.100588. eCollection 2021 Jun 18.


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).

Keywords: Microscopy; Molecular/Chemical Probes; Single-molecule Assays.