Fig. 2.

Our modified CO-FISH assay. a Schematic overview of our CO-FISH assay. A diploid cell with one pair of homologous chromosomes, containing Watson (W; blue) and Crick (C; red) strands is shown. BrdU present in the culture medium is incorporated into growing DNA chains during S-phase. The cell in G2 now contains four sister chromatids, each containing one strand of newly synthesized, BrdU-substituted DNA (indicated by zig–zag-shaped line). Two possibilities for sister chromatid segregation exist: (1) The WC:WC pattern is shown on top, where one old W- and one old C-template-strand-containing sister chromatid are segregated together to sister cells. (2) In the WW:CC pattern (bottom), sister chromatids of the same replication history are segregated together. One sister cell will contain both old W-template-strand-containing sister chromatids, and the other sister cell will contain both older C-template-strand sister chromatids. To simplify presentation, only one sister cell is shown for each segregation pattern. The next cell cycle is advanced in medium without BrdU, and cells are arrested in metaphase by colchicine treatment. After chromosome preparation and in situ strand degradation, directly labeled PNA probes complementary to sense (blue) and anti-sense (red) telomere repeat are hybridized to chromosomes. Different fluorescent signals from the single-stranded sister chromatid correspond to WC:WC segregation; whereas, identical fluorescent signals from the single-stranded sister chromatid correspond to WW:CC segregation. Because PNA probes invade double-stranded (ds) DNA, the ds telomere will show double fluorescent signals. Individual chromosomes are identified by additional hybridization of a chromosome-specific DNA probe. b Examples of metaphase spreads showing a WW:CC segregation pattern for chromsome 7 on the left, and a WC:WC segregation pattern on the right. DNA was stained with DAPI (blue), sense- and anti-sense telomere probes are shown in green and red. In situ identification of chr. 7 and major satellite repeat is shown in the bottom. White arrows help identify chromsome 7 in the top, and high-resolution images are placed in the top corners