Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, followed by isolating and stretching of genomic DNA. We then detail the detection of the replication at chromosomal regions through immunostaining and fluorescence in situ hybridization. Using SMARD, we can visualize replication initiation, progression, termination, and fork stalling.

For complete details on the use and execution of this protocol, please refer to Norio et al. (2001) and Gerhardt et al. (2014).^1,2