In this technique, glass cover slips are functionalized with a mixture of inert and biotin peg molecules. A flow cell is created with this functionalized surface and placed on a turf microscope. Biotinylated rolling circle templates are attached to the surface using multivalent streptavidin.
When the substrates are replicated by addition of repli zone proteins and nucleotides, the resulting double stranded DNA is extended with laminar flow and imaged in real time using an inter collating dye. Hi, I'm Nathan Tanner from Antoine Van Ian's lab in the Department of Biological Chemistry and Molecular pharmacology at Harvard Medical School. I'm Joe Laro also from the Van Noian lab.
Today we'll show you a procedure for fluorescence imaging of DNA replication in real time at the single molecule level. We use this procedure in our laboratory to study coordinated DNA synthesis activities of individual replication complexes. So let's get started.
To begin this procedure anil the biotinylated tail oligo to the M 13 single stranded DNA by adding a tenfold excess of oligo in TBS buffer heating to 65 degrees Celsius, then turning off the heat block to allow slow cooling to properly aneel the primer. Now that the prime M 13 is ready, add it to a mixture of 64 nanomolar T seven DNA polymerase and T seven replication buffer containing D NDPs and magnesium chloride and incubate the reaction at 37 degrees Celsius for 12 minutes. At the end of the incubation, quench with 100 millimolar EDTA.
Next purify the filled in product DNA with phenol isoamyl alcohol chloroform, extraction and dialyze into TE buffer or other suitable storage buffer back extract each organic phase to recover any residual primed. M 13 after dialysis determine the DNA concentration with UV vis spectrophotometer. One iteration of template preparation can make enough nanomolar concentration of template for hundreds to thousands of single molecule experiments.
Now that the DNA is ready proceed to preparing the functionalized cover slips. The DNA to be replicated is attached to glass cover slips functionalized with Amil Lane, the Oxy group of the Amil binds to the glass leaving an amin available for binding 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 by at inated 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. Begin by placing 20 to 25 milliliters of blocking buffer containing BSA in a desiccate to remove any air bubbles for later steps. Next, dilute 25 microliters of streptavidin in 100 microliters of PBS and spread the solution over the surface of a peg functionalized Cover slip incubate for 20 minutes at room temperature to allow the streptavidin to bind to surface biotin while preparing other parts of the chamber.
While the cover slip is incubating, cut a piece of double-sided tape with backing on both sides so that it matches the size of the quartz slide used to assemble the flow chamber. Mark the position of the tubing holes on the tape with a pencil so that an outline of the flow chamber can be drawn on the tape. After marking the holes make a two millimeter wide rectangle around the markings.
This rectangle will serve as the flow channel, so be sure to make straight sides and leave room around the tubing inlet and outlet. Now cut the marked tape along the drawn outline, making straight clean cuts to make 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 tubing for the inlet and outlet of the flow cell. It helps to 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 is finished. Rinse the strep avid ENC coated cover slip thoroughly with water and dry using compressed air. Be careful not to turn the cover slip over as only one side is functionalized.
Finally, 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 epoxy and let dry for a few minutes. When the epoxy seal is dry, begin blocking of the surface.
Using a 21 gauge needle. Pull some of the Degas blocking buffer through one of the tubes. Flush out a few times to remove air bubbles and allow the chamber to incubate for at least half an hour before moving on to the single molecule replication.Experiment.
Now that the flow cell has been blocked, place it on the microscope stage. Hold the chamber in place with stage clips and be sure that the flow channel is aligned along the y axis. Connect the flow cell outlet tube to the syringe pump using a larger diameter connector tubing.
Place the inlet in blocking buffer and pull back on the syringe to remove any air in the tube. A gentle flick of the outlet tube will help clear any air bubbles trapped in the flow cell. Now add the DNA, dilute the stock DNA template to 25 picomolar in one milliliter of blocking buffer flow into the chamber at moderate flow rate such as 0.025 milliliters per minute for 30 minutes to allow good surface coverage of DNA.
Once the DNA is in the chamber, wash out excess DNA using blocking buffer wash for at least 200 flow cell volumes. To get rid of all the free DNA turn on the laser and camera. The cooled CCD needs to reach temperature before it can be used.
After washing off excess DNA and degassing the replication buffer, prepare the replication reaction mix nucleotides DTT and cytoxin the replication buffer. Then add proteins and mix gently. Then flow the reaction mixture into the flow cell.
The flow speed here needs to be sufficient to stretch double stranded DNA for a two millimeter wide. 100 micrometer tall flow channel 0.04 milliliters per minute works well. Allow time for the mixture to enter the chamber about 10 minutes.
Move the field of view to the side of the channel and focus on the adhesive material to ensure one is near the surface for focusing and to begin imaging. Adjust laser power microscope focus and turf angle. Keep the power low to avoid any photo cleavage of the stained DNA.
Acquire at one to five frames per second for several minutes depending on the rate of DNA replication. Repeat to get longer replication trajectories or move to a new field to see more molecules when nearly all the reaction mixture has pumped into the cell flow in more buffer with cyt to see other fields of view. Taking multiple images of different replicated molecules provides statistics for productivity determination.
In the rolling circle. Replication assay leading strength synthesis extends the tail linking the circle and surface the tail is converted to double stranded DNA by the lagging strand polymerase and stretched by laminar flow shown. Here is an example field of view with cyt orange at 532 nanometer excitation.
The small fluorescent spots are tethered unreplicated substrates. Note the extreme length of the products and the large number of products per field. A typical field of view will show numerous replicating molecules visible as growing bright lines of stained DNAE Coli experiments at 37 degrees Celsius give five to 50 molecules per field depending on surface density.
The T seven repla zone initiates much more efficiently and will replicate more than 50%of the molecules in a field. With such a high density, it is difficult to resolve individual molecules as seen here. So perform the T seven experiment at lower protein concentrations or surface density Chime graphs of typical replicating molecules from e coli and T seven experiments include endpoint trajectories of molecules from e coli and T seven plotted versus time the trajectories are fitted with linear regression to obtain replication rates, which in this example are 467.1 base pairs per second for e coli and 99.4 base pairs per second.
For T seven, the the rate distributions of single molecule trajectories fit with single gaussians give a mean of 535.5 plus or minus 39 base pairs per second for e coli and 75.9 plus or minus 4.8 base pairs per second. For T seven, the length distributions of replication products are fit with single exponential decay to obtain productivity, which is 85.3 plus or minus 6.1 kilobase pairs for e coli and 25.3 plus or minus 1.7 kilobase pairs for G seven. Then We've just shown you how to measure coordinated DNA replication with single molecules.
When doing this procedure, it's important to remember to properly functionalize the cover slips and make clean straight flow channels. Be sure to keep the laser power load and minimize photo cleavage of the long DNA. So that's it.
Thanks for watching and good luck with your experiments.
