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

## TL;DR

This paper provides a detailed protocol for studying the replication of Kaposi’s sarcoma-associated herpesvirus using a technique called SMARD, which visualizes DNA replication at the single-molecule level.

## Contribution

The paper introduces a step-by-step protocol for using SMARD to investigate the replication kinetics of Kaposi’s sarcoma-associated herpesvirus.

## Key findings

- The protocol includes steps for nucleotide analog pulsing, agarose plug preparation, and fluorescence imaging to map replication origins.
- Instructions for isolating high-molecular-weight DNA and linearizing KSHV DNA using restriction digestion and PFGE are provided.
- The method allows for the detection of IdU and CldU in viral DNA through fluorescence microscopy.

## 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

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.