To begin, centrifuge one times 10 to the power of seven HeLa cell lines containing telomeric restriction fragments DNA at 1, 000g for three minutes. Discard the supernatant and resuspend the pellet in 200 microliters of PBS. After SDS proteinase K and sodium chloride treatment, centrifuge the cell suspension at 16, 900g for 10 minutes.
Transfer the supernatant to a new centrifuge tube and add an equal volume of isopropanol, avoiding floating lipids or sediment. Centrifuge at high speed, and wash the pellet with 500 microliters of 70%ethanol. After drying the DNA pellet, carefully resuspend it in 475 microliters of TE buffer and gently mix by tapping the bottom of the tube.
Now, add 25 microliters of 10 milligrams per milliliter RNAsay and tap the tube until the pellet is completely dissolved. Next, add 1/10 volume of three-molar sodium acetate and two volumes of cold 100%ethanol and place the tube at minus 80 degrees Celsius for two to three hours. After centrifugation and 70%ethanol washes, add 100 microliters of TE buffer to the DNA pellet, tap the tube to mix, and place it at four degrees Celsius for two hours to fully dissolve.
For DNA digestion, combine four micrograms of genomic DNA with one microliter of CviAII, two microliters of 10X digestion buffer, and water to reach a total volume of 20 microliters. Incubate the mixture at 25 degrees Celsius for 12 hours. In a thermal cycler, heat the mixture at 75 degrees Celsius for three minutes.
Then, decrease the temperature by 0.1 degrees Celsius every 30 seconds until reaching 25 degrees Celsius. After cleaning and drying, coat the bottom cover slips with 20 microliters of 0.1%nitrocellulose and add reference beads. Bake the cover slips at 100 degrees Celsius for four minutes.
Assemble the flow cell sandwich. And after heating at 85 degrees Celsius, use two swabs to massage the assembly until the Parafilm seals the channel. Wash 10 microliters of M-270 beads five times with 50 microliters of working buffer using a magnet.
After washing, add the beads on top of the digested genomic DNA into a 1.5-milliliter centrifuge tube. Gently flick the tube a few times to mix the beads and DNA sample. Leave the mixture on ice for one hour.
After incubation, wash the mixture with 500 microliters of working buffer three times using a magnet to pull the beads down with 10-minute intervals between washes. Resuspend the sample in a working buffer, and load 30 microliters of the mixture into the flow cell. Flush the unbound magnetic beads after 30 minutes.
After placing the flow cell on top of the objective lens, select a pair of five-millimeter cubic magnets arranged in a vertical configuration, and align the magnet holder with the x-axis of the magnetic tweezers'light path for imaging. Launch the graphical programming software and connect the controllers for the magnetic tweezers. Adjust the field of view to locate a reference bead at the bottom of the flow cell, and adjust the objective lens slightly so that the reference bead shows clear defraction rings.
Write a script in MATLAB to control motor movements for force ramp assays. Import the script into graphical programming software to test the single-molecule experiments. At a slow flow rate, load 200 microliters of 10-nanomolar TRF1 into the flow cell.
After 30 minutes of binding, choose a script for a force ramp experiment with a force loading rate of plus/minus one piconewton per second. Name the data files and run the experiment. Genomic DNA integrity was confirmed using agarose gel electrophoresis, with the resulting TRS displaying consistent lengths across various human cell lines.
Telomeric repeat sequences in TRS were detected via southern blotting, showing clear hybridization signals. Force extension curves from the force ramp assay showed zigzag patterns during stretching, indicating the breaking of protein DNA interactions. The dissociation kinetics of telomeric DNA protein complexes showed a linear relationship between force and dissociation rate.
Moreover, the length heterogeneity of telomeric DNA from human cells investigates the loop formation mechanism in telomeres of various lengths.
