Figure 2. Combing full-length fission yeast chromosomes.

Fission yeast genomic DNA was labelled in vivo with BrdU using a synchronous cell population. Genomic DNA was prepared for DNA combing from cells at mid S-phase. Single DNA molecules immobilized on glass slide were denatured and BrdU incorporated into newly synthesised DNA was visualised with fluorescently labelled anti-BrdU antibody (green). The whole DNA molecule was counterstained with anti-single stranded DNA antibody (red). (a) A representative DNA molecule completely replicated in the presence of BrdU is shown. The quantification of false negative staining, corresponding to the gaps in the BrdU signal expressed as number of pixels is shown below the molecules. Ten DNA molecules corresponding to 5Mb of DNA were analysed. We used a gap size threshold of ≤10 pixels to identify false negative staining in the segmentation of BrdU tracks. (b) A representative unlabelled DNA molecule stained with anti-single stranded DNA antibody (red) and anti-BrdU antibody (green) is shown. The quantification of false positive staining, corresponding to green dots on the molecules expressed as number of pixels is shown below the molecule. Ten DNA molecules corresponding to 7 Mb of DNA were analysed. We used a threshold of >3 pixels to identify replication tracks. (c) An example of DNA molecules undergoing DNA replication is shown. White arrows point to the middle of replication tracks (shown with a short white line below the DNA molecule) where replication origins are most likely located. The inter origin distance represents the distance between the middle of two adjacent tracks. Biotinylated FISH (fluorescent in situ hybridisation) probes (shown in blue below the molecule) were used to align DNA molecules to their corresponding chromosomal sequences. DNA probes were designed with unique signature left and right arms and a gap between them, allowing the simultaneous detection of different chromosomal loci.