תצפית ישירה של ה-DNA אנזימים שכפול באמצעות DNA מולקולה בודדת מתיחה Assay

This video demonstrates a protocol for visualizing DNA replication by widefield optical microscopy linearized and chemically modified lambda. DNA is attached to a functionalized glass cover slip. The free end of the DNA is bead labeled and the DNA is stretched by laminar flow.

When replication is initiated, the leading strand begins to lengthen and the lagging strand shortens due to coiling. The large difference in length between double stranded and single stranded DNA at low forces allows a direct observation of DNA conversions during replication. The length of the DNA construct is monitored by tracking the position of the bead, which allows precise determination of the rate and process of DNA replication.

Hi, I'm Ari Ulrich from Laboratories of Anto one Vanian and Charles Richardson in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. Today we are going to show you a procedure for imaging DNA replication at single molecular level. We use this procedure in our laboratories to study enzymatic activities that take place during DNA synthesis.

So let's get started At the start of this procedure. Linearized bacteria phage Lambda DNA is modified by a kneeling oligonucleotides to form a replication fork, a lambda complementary fork arm, biotinylated fork arm, and a fork primer. The biotinylated fork forearm contains a biotin t attached to its five prime end, which allows tethering of the DNA template to the surface of the flow cell.

The lambda DNA is further modified by annealing, another oligonucleotide that contains a diggen and moiety serving as a high affinity immuno tag for attachment of a bead. To generate the replication fork, obtain lambda DNA in addition to several oligonucleotides whose sequences can be found in the written protocol accompanying this video. These oligonucleotides include a biotinylated fork arm, a lambda complementary fork arm, a fork primer, and a lambda complementary diggen and end phosphorylate the five prime ends of the lambda complementary fork arm and the lambda complementary digo genin end using T four polynucleotide kinase.

Next mix the phosphorylated lambda complementary fork arm, the biotinylated fork arm, the fork primer, and the lambda DNA in the molar ratio of 10 to 10 to 100 to one. In T four DNA Ligase buffer heat the mixture at 65 degrees Celsius for five minutes and slowly cool to room temperature by turning off the heat block to allow proper annealing of the oligonucleotides and T four DNA ligase to the mixture and incubate for two hours. At room temperature, T four DNA Ligase will catalyze sealing of the nicks between the oligonucleotides and the lambda.

DNA finally anil the lambda complementary diggen and end to the DNA replication template by adding the phosphorylated lambda complementary diggen and end in T four Ligase buffer incubate the mixture for 30 minutes at 45 degrees Celsius and again, slowly cool to room temperature by turning off the heat block ligate the phosphorylated Lambda complementary dig oxygen and end to the DNA replication template by adding T four DNA ligase to the mixture and incubate for two hours at room temperature. The final DNA replication template is now ready for use. In order to attach the tassel activated magnetic beads to the DNA replication template, they must be functionalized with an FAB fragment specific for digo genin.

To begin this process, resuspend the stock solution of beads and transfer 400 microliters into an einor tube. Place the tube into a magnetic separator and incubate until the solution clears. Then remove the supernatant by pipetting To activate the toal groups for antibody coupling, add one milliliter of boid buffer and mix gently by pipetting.

Again, remove the supernatant with the aid of a magnetic separator. Next, add 240 microliters of FAB fragment and resuspend the beads in 400 microliters of borate buffer. Mix thoroughly and allow incubation on a rotator for 16 to 24 hours at 37 degrees Celsius.

After incubation, separate the beads using the magnetic separator and remove the supernatant. Wash the beads with one milliliter of washing buffer and incubate for five minutes at four degrees Celsius, remove the buffer using the magnetic separator. This wash is repeated twice.

To block the free tosil groups, add one milliliter of BSA containing tris buffer and incubate for four hours at 37 degrees Celsius. Following incubation, remove the buffer from the beads using a magnetic separator wash twice with buffer and aliquot. For use, the DNA to be replicated is attached to glass cover slips functionalized with Amil lane.

The oxy group of the Amil lane binds to the glass leaving an amin available for binding to biotinylated peg molecules. This coating helps reduce non-specific binding to the surface. To thoroughly clean the glass cover slips, place them in staining jars and fill the jars with ethanol sonicate.

For 30 minutes, rinse out the jars and fill with one molar potassium hydroxide sonicate for 30 minutes and repeat both washes. For the first functionalization reaction, all traces of water need to be removed. Fill the staining jars with acetone and sonicate for 10 minutes.

Empty the jars and fill again with acetone. Proceed to add the sine reagent to the jars and agitate for two minutes. Then quench with a large excess of water.

Then dry the cover slips by baking them at 110 degrees Celsius in the oven for 30 minutes. Next, prepare the peg mixture at about 0.2%weight per volume. Biotinylated peg, including a glass cover slip spacer, will allow separation of the cover slips, pipette 100 microliters onto a dry siloized cover slip.

Place a second cover slip on top. Incubate the cover slips in the PEG solution for three hours. Then separate each pair of cover slips and wash extensively with water.

Be careful to keep the cover slips functionalized side up as only one side will be coated with peg. Finally, dry the cover slips with compressed air and store them under vacuum In a desiccate, the surfaces remain stable for at least a month. So dozens of cover slips can be made in a batch and used as needed.

Now that the cover slips are ready, one can assemble the flow chamber for the single molecule experiment. The single molecule experiment is performed using a simple flow chamber constructed with a functionalized cover slip, double-sided tape, a quart slide and tubing. One flow chamber is prepared for each single molecule experiment.

Begin by mixing five milliliters of blocking buffer and 20 milliliters of working buffer and placing the mixture in a desiccate to remove any air bubbles for later steps. Next, spread 100 microliters of a working solution of strp din in PBS over the surface of the peg functionalized Cover slip incubate for 20 minutes at room temperature to allow strp din to bind surface biotin. Meanwhile, cut a piece of double-sided tape to match the size of the quartz slide mark the position of the tubing holes on the tape.

Outline the flow chamber by drawing a three millimeter wide center channel and rectangles containing both holes. Flow chambers can be designed with two inlet and two outlet y shaped channels. Now cut the marked tape along the drawn outline, making straight clean cuts to be sure no adhesive protrudes into the channel.

Clean the quartz slide thoroughly using acetone to remove adhesive from flow cell construction used in previous experiments. Find the best alignment of the channel outline. Remove one side of the adhesive backing and carefully place the tape onto the court slide.

Be careful to align the tape properly as the inlet and outlet holes need to remain unblocked. Next, prepare the tubing cut lengths of 0.76 millimeter inner diameter tubing for the inlet and outlet of the flow cell. Cut the end of the tube at about a 30 degree angle so that the tube will not press flat against the chamber bottom.

Suspend the tubing on test tube racks for easy attachment to the flow cell in the next steps. By now, the cover slip incubation should be finished. Rinse the cover, slip thoroughly with water and dry it using compressed air.

Be careful not to turn the cover slip over as only one side is functionalized with peg. Complete the assembly of the flow chamber by removing the other side of the tape backing and placing the quartz slide onto the cover slip Lightly press on the cover slip to push out any air trapped in the adhesive. This will help prevent any air bubbles getting into the flow channel.

Seal the sides of the chamber with epoxy. Insert the cut tubing into the holes of the quartz and seal in place with the epoxy. Let dry for a few minutes.

Once dry, use a 21 gauge needle to pull some of the DGAs blocking buffer through one of the tubes in order to block the chamber surface. Flush out a few times to remove air bubbles and allow the chamber to incubate for at least 30 minutes before moving onto the single molecule replication experiment. Once the DNA template functionalized beads and the flow chamber have been prepared, a single molecule experiment can be performed.

To begin, place the flow cell on the microscope stage. Hold the chamber in place with stage clips and be sure that the flow channel is positioned in alignment with the CCD camera. Connect to previously constructed air springing to the syringe pump and to the outlet tubes of the flow cell.

Using larger diameter connector tubing, place the inlet tubes of the flow cell in blocking buffer and pull back on the syringe to remove any air bubbles from the tubes, gently flick the outlet tubes to clear any air bubbles trapped in the flow cell. Once all air bubbles have been removed, plug one of the inlet tubes with a needle and seal one of the outlet tubes by bending it 180 degrees and securing with adhesive tape Prior to adding DNA to the cell, dilute the stock DNA template to 10 picomolar in one milliliter of DGAs one x blocking buffer. Flow the template into the chamber at a moderate flow rate to allow good surface coverage of the DNA.

Dilute the stock bead solution to between two and four times 10 of the sixth beads per milliliter. In one milliliter of DGAs one x blocking buffer. Then vortex the mixture and sonicate for 20 seconds.

Flow the diluted bead solution into the chamber. Once beads are added, turn the flow off and allow the chamber to reach equilibrium. Then close both outlet tubes.

Any changes to the tubes without the chamber closed will cause pressure fluctuations that will exert a strong force on the beads and shear the tethered DNA molecules. Plug the inlet tube that was used for loading the beads with a needle using the second inlet tube. Begin washing the flow cell with DGAs one x solution of blocking buffer wash extensively.

Agitate the stage by tapping to remove any beads that are non-specifically stuck to the surface to check it. The beads are tethered to the DNA. Try changing direction of the flow.

Once free beads are sufficiently removed, turn the flow off and allow the chamber to reach equilibrium. Prepare fresh reaction mixture containing replication proteins as indicated in the text protocol. Close the open outlet tube and switch the inlet reservoir to the freshly prepared reaction mixture.

Slowly opening the outlet to avoid rapid pressure changes to proceed with the enzymatic reaction. First, flow the protein solution into the chamber at a moderate flow rate to allow efficient binding to the DNA. Turn off the flow and allow the chamber to reach equilibrium.

Close the outlet tubes and replace the tube containing the reaction mixture. With the tube consisting of DGAs washing buffer, wash the chamber to remove free proteins after washing. Again, turn off the flow and allow the chamber to reach equilibrium.

Close the outlet tubes and replace the tube containing washing buffer. With the tube containing the T seven replication buffer, the T seven replication buffer contains magnesium chloride that triggers the start of the replication reaction. Use a flow rate of 0.012 milliliters per minute that corresponds to three pico Newin drag force on the DNA tethers using conditions described in this protocol.

Place a rare earth permanent magnet above the flow cell before imaging to prevent non-specific interactions between the beads and surface. Position a fiber illuminator at an incidence angle between 10 degrees and parallel to the microscope stage near the microscope view field with a 10 x objective and CCD camera. Dark field illumination is used to increase the contrast in the bead imaging focus using a tethered bead.

Begin data acquisition acquire for 20 to 30 minutes at a slow frame rate. In a successful experiment, 100 beads can be simultaneously observed. The experiment should yield numerous traces displaying leading strand DNA synthesis.

In order to measure rates and processive of DNA unwinding and polymerization, the precise positions of beads must be determined to achieve this. Fit these positions with two dimensional Gaussian functions. Visualize the trajectory by tracking the bead position in each frame using tracking software.

Then plot bead position as a function of time using any graphic software to obtain rate data. Fit the plots with a linear regression and calculate the slope for processive. Determine the total length of the DNA from start to end of a shortening event.

Combine all single measurements and plot rate and productivity distributions fit the data using Gaussian and single exponential functions respectively. We've just shown you how to measure rates and processive of DNA replication on a single molecule level. When doing this procedure, it's important to make sure that the force and certain of bets by the lamina flow doesn't affect enzymatic activities of the replication proteins.

So that's it. Thanks for watching and have fun on your experiments.