Exponentially growing cells in culture are sequentially pulsed with two different halogenated nucleosides. DNA replicating within these pulse periods becomes labeled with the corresponding nucleoside. Pulsed cells are embedded in agarose plugs and lysed, and remaining embedded genomic DNA is digested with a rare cutting restriction endonuclease to produce segments of 100 – 600 kb. The digested DNA is then separated according to size by pulse field gel electrophoresis and the target segment within the gel is identified by Southern blotting. A gel slice containing the target segment is excised from the gel and melted. The DNA in the melted gel solution is then stretched on silanized glass slides and the halogenated nucleosides incorporated in the replicated DNA are detected by immunostaining. Biotinylated FISH probes are used to identify the target molecules and to align the images of individual molecules to produce a composite profile of replication. Events that can be detected include: replication forks (white arrows) moving in the 5’-3’ (I) and 3’–5’ (II) directions; initiations (III); and terminations (IV).
Since all the labeling is carried out under physiological conditions, the results of SMARD directly reflect the replication process in vivo. This allows us to determine how DNA replication initiates, progresses, pauses and terminates throughout the genomic region analyzed. SMARD has many advantages: it does not require synchronization of the cells; it can simultaneously map replication initiation and termination sites, determine fork direction, measure the average rate of fork movement, and identify strong pausing sites; and it requires fewer than 107 cells, which makes the study of DNA replication possible in primary cells.
Recently we have optimized the methodology to allow us to perform SMARD on megabase-sized DNA segments. This not only expands our ability to study replication in large chromosomal regions from mammalian cells, but also allow us to efficiently study replication origins of prokaryotic organisms. We are exploring more applications and collaborations for this recently developed technique. Meanwhile we are trying to improve the SMARD technique to make it amenable to automatic processing.