Profiling of DNA replication timing in unsynchronized cell populations

Profiling chromatin in a particular cell type provides a valuable 'signature' for cell identity and developmental stage. One approach has been to assay and use the timing of DNA replication across a panel of loci as an indicator of chromatin structure. This epigenetic profiling used on pluripotent embryonic stem (ES) cells has reliably distinguished them from cells that have a more restricted lineage potential. Thus, such an approach may become increasingly useful for understanding the molecular basis of pluripotency and lineage induction, especially in the context of stem-cell therapy. Here I describe in detail the DNA replication timing method, whereby unsynchronized cell populations are pulse-labeled with 5-bromo-2 '-deoxyuridine (BrdU), fractionated according to cell-cycle stage and the abundance of candidate sequences within newly replicated DNA is determined by PCR. This robust protocol has been used consistently by several laboratories and might offer some advantages over conventional transcription-based profiling for characterizing cell populations. The procedure requires 3-4 d to complete.