Identification of late DNA-replicating regions in Chinese hamster chromosomes using a BrdU-Giemsa technique

Asynchronous Chinese hamster cells were labelled with BrdU for 3 h prior to harvesting the metaphase cells. The late DNA replicating sites became unifilarly BrdU-substituted as compared to the earlier replicating sites having a normal DNA constitution. Those late replicating sites were identified by pale coloration or dot formation after treatment with 1.0 M Na-phosphate solution (adjusted to pH 9.0 with supersaturated amount of NaHCO3 and at a temperature of 69-75 degrees C) and staining with Giemsa dye. Using this technique, nuclei with incorporated BrdU could be distinguished from nuclei that had not incorporated BrdU. --One of the advantages of using this technique for identification of late DNA replicating sites is that cells are treated continuously with BrdU for a short period of time before harvesting and only one sampling, rather than a series of samplings, is required to achieve a clear-cut result.