Protocol to investigate replication kinetics of Kaposi’s sarcoma-associated herpesvirus using single-molecule analysis of replicated DNA

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.