Protocol to investigate replication kinetics of Kaposi’s sarcoma-associated herpesvirus using single-molecule analysis of replicated DNA
Year: 2025
Type: article
Abstract: Single-molecule analysis of replicated DNA (SMARD) is a powerful tool to study DNA replication by visualizing de novo -incorporated nucleotide analogs in individual DNA molecules. Here, we present the protocol to investigate replication kinetics of Kaposi’s sarcoma-associated herpesvirus using SMARD. We describe steps for seeding cells, nucleotide analog pulsing of cells, preparation and digestion of agarose plugs followed by pulsed-field gel electrophoresis, probe labeling, DNA stretching, and fluorescence imaging to map replication origins and fork progression. For complete details on the use and execution of this protocol, please refer to Verma et al. 1 and Singh et al. 2 • Steps for pulsing cells with sequential nucleotide analogs for replication labeling • Procedures for embedding cells in agarose plugs and isolating high-molecular-weight DNA • Instructions for linearizing KSHV DNA using restriction digestion and PFGE • Guidance on stretching viral DNA and detecting IdU and CldU by fluorescence microscopy Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Single-molecule analysis of replicated DNA (SMARD) is a powerful tool to study DNA replication by visualizing de novo -incorporated nucleotide analogs in individual DNA molecules. Here, we present the protocol to investigate replication kinetics of Kaposi’s sarcoma-associated herpesvirus using SMARD. We describe steps for seeding cells, nucleotide analog pulsing of cells, preparation and digestion of agarose plugs followed by pulse field gel electrophoresis, probe labeling, DNA stretching, and fluorescence imaging to map replication origins and fork progression.
Source: STAR Protocols
Institution: University of Pennsylvania
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W7116657579