Molecular Combing of Single DNA Molecules on the 10 Megabase Scale

DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions.

(a) Fission yeast cells were synchronized at the G2/M transition by blocking cdc25ts cells at 36°C followed by temperature shift to 25°C, after which cells proceeded synchronously through mitosis and G1 into S-phase. 2µM BrdU was added to the synchronous cultures at G1 allowing cells to incorporate BrdU into newly synthesized DNA upon entry into S-phase. We have established that DNA synthesis initiates around 45 minutes after release from the temperature block by labeling a cdc25ts synchronous cell population with (3H)BrdU at 5 minute intervals and measuring the radioactivity in purified genomic DNA for each time point (data not shown). DNA was prepared for DNA combing from cells 75 minutes after release, corresponding to mid S-phase (estimated from FACS profiles, and (3H)BrdU labeling).
(b) FACS profile of synchronously progressing cdc25ts cell population. The numbers on the left side of the FACS profile indicate the time points after release from the temperature block. The time points corresponding to S-phase are indicated with a red line and the time of the BrdU pulse in G1 is indicated with an arrow. Fission yeast cells undergo DNA synthesis before completing cytokinesis, which explains the FACS profile. The peak of septation was estimated to be 70%. L < 1 6 1 6 < L < 3 2 3 2 < L < 6 4 6 4 < L < 1 2 8 1 2 8 < L < 2 5 6 2 5 6 < L < 5 1 2 5 1 2 < L < 1 0 2 4 1 0 2 4 < L Supplementary Figure S3. Scheme for in vivo DNA labelling of U2OS cells.
(a) An asynchronous cell population was blocked at the beginning of S-phase using 2.5mM thymidine and then released into 100ng/mL nocodazole to arrest cells in mitosis. Then cells were released from the mitotic block and BrdU was added to the culture after 5 hours (corresponding to G1, see FACS profile below) which allowed cells to incorporate BrdU into newly synthesised DNA upon entry into S-phase. DNA was prepared for DNA combing from cells at 12 hours after release, corresponding to mid S-phase (estimated from FACS profiles).
(b) FACS profile of synchronously progressing U2OS cell population. At release (t=0h) cells were blocked at mitosis and progressed synchronously through mitosis, G1 and S-phase. 10µM BrdU was added 5 hours after release (during G1) allowing cells to incorporate BrdU into newly synthesized DNA upon entry into S-phase. DNA was prepared for DNA combing from cells at 12h after release, during S-phase (estimated from FACS profiles).
(c) To estimate the fraction of cells in early, mid and late S-phase, we pulsed labeled synchronous cell population at the indicated time points with 10µM BrdU for 10 minutes. Cells were ethanol fixed and stained with anti-BrdU antibody and propidium iodite in order to measure the DNA content. We plotted the DNA content on the x-axis and the BrdU intensity on the y-axis. We found that 10h, 12h and 14h after release correspond to early, mid, and late S-phase respectively. (c) Plot of the minimum number of origins fired within 40kb of each other, for molecules replicated up to 50% (45Mb analysed). The data points lay on plot with a descending segment reaching saturation at 6 origins per cluster. This suggests that 6 fired origins is the minimum number of origins defining a cluster in U2OS cells.
Assuming that a minimum of 6 fired origins define a cluster, cells having replicated 50% of their DNA will have on average 3200 clusters.
Minimum origins per cluster Number of clusters per genome (Seikagaku) and 0.3µg/µL Lysing Enzymes (Sigma) at 37°C. We added equal volume of 2% low melting agarose Mb grade (BioRad) and 0,2% NaN 3 melted in SP1 and equilibrated at 45°C, such that each agarose plug (80µL) contained 1.5-2x10 7 of fission yeast cells. The cell suspension was mixed gently and thoroughly, added to plug molds (BioRad) and incubated at 37°C until 100% "ghost" formation (tested with 10% SDS).
Agarose blocks were placed at 4°C for 10 minutes to solidify, ejected in Digestion Buffer 2 (1% N-Laurouylsarcosine, 1mg/ml Proteinase K, 125mM EDTA pH 9.5) and incubated for 1 hour at 50°C. The DB was changed and the plugs were incubated in fresh DB for 48 hours at 50°C, changing the buffer twice more. The plugs were washed for two days in TE 1X pH 7.5 with 100mM NaCl (buffer changes) and melted in MES 50mM pH 6 with 100mM NaCl within combing reservoirs for 15 minutes at 70°C. Combing reservoirs were cooled down to 42°C before adding 2µl β agarase (New England BioLabs) without mixing and incubated overnight at 42°C. Combing reservoirs were cooled down to RT and genomic DNA was combed onto silanised glass surfaces using the combing machine at a speed of 900µm/sec. DNA was dehydrated by incubating slides 2 hours at 65°C and stored at -20°C.
The DB was changed and the plugs were incubated at 50°C for 48 hours, changing the buffer twice more. The plugs were washed for two days in TE 1X pH 7.5 with 100mM NaCl before melting them in combing reservoirs and adding 2µl β agarase (New England BioLabs) without mixing and incubated overnight at 42°C.

FISH Probe Hybridisation and Immunodetection. Glass slides with combed DNA
were incubated in 1M NaOH for 30minutes in a coplin jar to denature DNA molecules. Finally, running the executable requires a formatted input file, e.g. "data.csv", saved in the working directory. To run the executable on a Unix machine, enter the following in the command line: ./makeBarcode.exe data.csv barcode.eps 8 22