Figure 1. Replication bands. (a) Replication bands on human chromosomes. Human cells were incubated with BrdU for 5.5 h before harvesting, resulting in labeling of late-replicating regions. Chromosomes were prepared and stained for replication bands. Light bands indicate areas that incorporated BrdU. The inactive late-replicating X chromosome is marked by an arrow (see text) (From Verma RS and Babu A (1995) Human Chromosomes: Principles and Techniques. New York, NY: McGraw-Hill, reprinted by permission from McGraw-Hill, Inc.) (b) Replication bands after continuous labeling with BrdU. In this scheme, the regions that incorporated BrdU appear as dark bands. The pattern of replication bands varies according to the length of the incubation period with BrdU before harvest. The arrows point to the position in the cell cycle at which the chromosome was exposed to BrdU. The chromosome labeled for 1 h lacks replication bands because the BrdU incubation period initiated when the chromosome was in G₂, and had already completed DNA replication. However a 3 h incubation incorporated BrdU at the end of S phase, therefore the bands represent the latest replicating regions. As the incubation period lengthens, the chromosome is covered with more and more replication bands. For an incubation period that exceeds the sum of G₂ and S phases, the entire chromosome replicated in the presence of BrdU and is therefore stained uniformly.

Figure 2. Fluorescent in situ hybridization (FISH) replication timing assay. (a) Patterns of hybridization representing nonreplicated and replicated regions. The nucleus on the left represents a BrdU-positive cell (in S phase), indicated by its staining (by 7-amino-4-methylcoumarin-3-acetic acid (AMCA)), hybridizing with a singlet–singlet pattern. Each hybridization dot represents one chromosomal copy that has not yet replicated. The nucleus on the right represents a BrdU-positive cell with a doublet–doublet pattern, each doublet representing a pair of sister chromatids originating from one chromosome. The presence of a doublet indicates that the region of interest has replicated. (Figure courtesy of Rachel Ofir.) (b) A schematic explanation for the FISH replication timing assay. Distribution of the hybridization patterns relative to the position of the cells in the cell cycle is indicated in the figure. Nuclei from G₁ hybridize with a singlet–singlet pattern, while nuclei in G₂ hybridize with a doublet–doublet pattern. In metaphase, two signals emanate from each chromosome. In S-phase nuclei, the pattern of hybridization depends on the timing of replication of the region of interest. All nuclei from cells positioned in S prior to the point where replication takes place (shown by an arrow) will show the singlet–singlet pattern, while nuclei originating from cells positioned after the point of replication will display the doublet–doublet pattern. Thus the timing of replication will affect the percentage of nuclei showing either of the hybridization patterns. (Modified version of an illustration by Asaf Hellman.)

Figure 3. Replication timing analysis by cell elutriation. Cells from the human erythroleukemia cell line K562 were labeled with BrdU and separated to five fractions in S phase (labeled S1–S5, from early to late in S phase) by elutriation. Southern blot hybridization was performed on BrdU–DNA isolated from the different fractions and digested with EcoRI. Different probes give different patterns of hybridization. A probe from the human -globin gene, active in this cell line, hybridizes preferentially to BrdU–DNA that replicates early in S phase, while a probe from the region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is inactive in these cells, hybridizes to BrdU–DNA replicating late in S phase.