The overall goal of this procedure is to visualize DNA replication on the single molecule level. Begin by incorporating halogenated nucleotide analogs into newly synthesized DNA. In living cells spot the cells with labeled DNA onto a microscope.
Slide then lies the cells and stretch out the DNA onto the microscope slide. Subsequent immuno staining of DNA fibers and analysis of the fluorescent microscopy images can illuminate the global replication fork dynamics to allow quantitative analysis of parameters that influence the overall replication program. Over the past years, different version of the DNA fiber Fluor techniques were developed to visualize the movement of individual replication fork within living cells.
This in vivo experiment in DT 40 cells that you're just about to witness can be accomplished within a day and requires only general laboratory equipment and a fluorescent microscope demonstrating the procedure will be Dr.Rebecca Schwab, a postdoc for my laboratory. In order to label DT 40 cells in vivo, start with an exponentially growing culture. Add IDU label to a final concentration of 25 micromolar and mix the cell suspension.
Well incubate the cells for 20 minutes at 38 degrees Celsius and 5%carbon dioxide. Next, add the CLDU label to a final concentration of 250 micromolar. Mix and incubate the cell suspension.
Now wash the DT 40 cells with ice cold PBS reus. Suspend the cell pellets in cold PBS and keep the labeled cells on ice. Spot two microliters of the cell suspension onto one end of the glass slide air dry until the volume of the drop is greatly reduced but not completely dry.
Now, add seven microliters of fiber lysis solution on top of the cell suspension, and gently mix the solutions by stirring with a pipette tip incubate for two minutes. Next, tilt the slides to 15 degrees to allow the fibers to spread along the slide. Once the fiber solution has reached the bottom of the slide, place it horizontally to air dry.
At this point, a thin opaque line should be visible along the slide. Mark the beginning of the stretched fibers with a pencil. First immerse slides in three to one methanol to acetic acid for 10 minutes.
Wash the slides in distilled water, then immerse in 2.5 molar hydrochloric acid for 80 minutes. Wash the slides three times in PBS for five minutes dab. Collect excess PBS onto a paper towel.
Then orient the slides horizontally. Now pipette 5%BSA in PBS on top of each slide, and place a cover slip incubate for 20 minutes. Move the cover slip gently down the glass slide.
Remove the excess BSA with a paper towel. Then pipette 50 microliters of the anti BRDU primary antibody solution. Onto each slide.
Cover a game with a cover slip and incubate in a humidified chamber for two hours. After removing the cover slips, wash the slides three times in PBS for five minutes. Apply 50 microliters of the secondary antibody solution position cover slips, and also protect the slides from light.
Then incubate for one hour. Remove the cover slips and wash the slides three times in PBS for five minutes. Finally, add a drop of vector shield mounting medium onto each slide.
Gently press on a cover slip and remove excess fluid around it. With a paper towel, seal the cover slips with transparent nail polish and air dry. Store the slides at minus 20 degrees Celsius.
Place a drop of immersion oil onto a slide close to the pencil mark and start locating the fibers. Move away from the main bundle to find areas where fibers are clearly separated from each other. In a typical experiment, select pictures only using one color channel in order to avoid bias.
Then take approximately 10 pictures of each sample. Move along the slide to take the different pictures as one area of a slide may not provide representative fiber lengths or replication structures. Import pictures into an image analysis program.
Measure the lengths of the 100 fiber tracts and or count 150 to 200 different replication structures. The double labeling fiber technique allows one to distinguish between different replication structures. Newly replicated DNA can be visualized as lines of antibody labeled nucleotide analogs.
Here, an ongoing elongating fork is represented as adjacent red and green signals. New initiation events can be divided into origins that have fired while the cells were incubated with the first label and origins that fired during the incubation with the second label. The former consists of neighboring green, red green signals and the latter of a green line.Only.
Termination events manifest as adjoining red green. Red signals interspersed. Origins consist of consecutive origins and termination signals.
Depending on the experimental design, stalled or collapsed forks can either be defined as a red only signal or a red line, followed by a short green tract. In this experiment, the wild type DT 40 cells have an average fork speed of 0.4 microns per minute. With 63%ongoing forks, 10%origins, 16%stalled forks, 8%terminations, and 3%interspersed fibers.
After watching this video, you should have a good understanding of how to visualize and analyze replication fork dynamics in DT 40 cells. This will provide you with an essential tool for investigating defects in DNA replication.
