Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

This protocol can be applied to any process that results in double stranded DNA breaks, yields detailed quantitative data on kinetics. Although this method has single molecule resolution, it measures up to 1000 DNAs in a single experiment, thus providing good statistics. The primary application of this method is to study the mechanisms involved in DNA binding and modification.

For example, we are interested in studying DNA target search, which is relevant to all DNA binding proteins. When attempting this technique for the first time, remember that single molecule tethers are delicate, and once form, must be handled with care. Proper proportion of functionalized surfaces is essential.

There are a few tricks for making the flow so. As well as switching sample tubes during data collection, visual demonstration can help someone who is new to this procedure. To wash the cover slips, place them in staining jars and sonicate them with ethanol for 30 minutes.

Thoroughly rinse them with deionized and distilled water, then immerse them in one molar potassium hydroxide and sonicate them for 30 more minutes. Repeat the wash sequence, making sure to rinse the cover slips with water after each wash. Store the cleaned cover slips in staining jars with water.

Use a clean razor to cut eight centimeter long loading and exit tubes, and insert them into holes in a clean glass slide. Epoxy the tubing and let cure for five minutes to secure it. Apply double sided tape pre-cut with the channel pattern to the glass slides, and smooth it out with plastic forceps to achieve a good seal.

Peel the backing off the tape, and apply a clean cover slip, smoothing it out with the forceps. Then, epoxy the edge of the cover slip to seal the flow cell and let it cure. Prepare the labeled DNA for tethering by performing PCR according to manuscript directions.

Then purify the PCR product with a PCR cleanup kit. Prepare 10 milliliters of buffer A, and degas it in a vacuum desiccator for at least one hour. To functionalize the flow cell, inject 25 microliters of anti-digoxigenin and fab fragments in PBS, into the flow channel, using a gel loading tip to fit into the PE60 tubing.

Incubate the flow cell at room temperature for 30 minutes. After the incubation, pull 0.5 milliliters of buffer A through the channel with a syringe, taking care to not introduce air into the channel. Then, mountain the flow cell on an inverted microscope.

Hook up the outlet tube to a syringe pump, and put the inlet tube into a micro centrifuge tube containing buffer A.Manually pull at least 0.5 milliliters of buffer A to flush the system and prime the pump. Then let the pump run at 10 microliters per minute for at least five minutes to equilibrate the system. Vortex the stock bottle of beads, and pipette 1.6 microliters of the beads into 50 microliters of buffer A, then vortex them again.

Place the beads on a magnetic separator and pipette out the buffer. Re-suspend them in 50 microliters of buffer A and vortex them. After the last wash, re-suspend the beads in 100 microliters of buffer A for a final concentration of 160 micrograms per milliliter.

Prepare 480 microliters of 0.5 picomolar labeled DNA substrate in buffer A, and pipette 20 microliters of bead suspension into the diluted DNA. Place the mixture on a rotator for three minutes. After the three minutes, immediately load the sample into the channel at a flow rate of 10 microliters per minute for 15 minutes, or until sufficient bead tethering is observed.

Wash the channel of all free beads by switching the inlet tube to a fresh tube of buffer A, and flowing it in at 50 microliters per minute for at least 10 minutes or until no loose beads are observed. And it's helpful to use an inline valve to cut off flow and switching the tubing. Switching tubing in one smooth motion and make sure liquid levels in new and old sample tubes match to avoid backflow.

Before collecting data, prepare the correct concentration of DNA cleavage enzyme, and insert the inlet tube into the sample. Lower the magnet over the flow cell. Set the pump to inject 80 microliters of sample at 150 microliters per minute and begin data collection.

One minute into data collection, activate the pump. Shown here is a representative image of the tethered beads before and after the reaction. Data analysis was performed to quantify the number of beads remaining tethered throughout the cleavage reaction.

This technique was used to measure the site specific DNA cleavage rates of Nde1 for protein concentrations ranging from 0.25 to four units per milliliter, and two different concentrations of magnesium. Each condition was replicated at least twice, with a few 100 to 1000 tethered DNA is per experiment. At sufficiently low protein concentrations, the rate is proportional to protein and independent of magnesium.

For high protein concentrations, the rate is dependent on magnesium, but independent of protein concentration. When attempting this protocol, keep in mind that air bubbles in the tubing and flow channel can run the experiment. Make sure not to allow any air into the lines while switching tubing, and degas all buffers before using.

The tethering method allows one to explore the effect of tension in the DNA on the reaction. This can be achieved by varying the magnet strength or applying flow during data collection.