The overall goal of this procedure is to examine the kinetics of primer and lagging strand DNA synthesis. This method can help answer key questions in the DNA replication field, such as how primers form by DNA primase, are transferred to, and utilized by, DNA polymerase. The main advantage of this technique is that it allows the identification of key steps during Okazaki fragment synthesis.
Since radioactive materials are used in this experiment, all institutional regulations regarding the safe use and disposal of radioactive materials must be followed. Start by aliquoting two microliters of formamide dye buffer into eight 1.5 milliliter polypropylene tubes, equal to the number of time points to be analyzed. Next, prepare a 20 microliter mixture consisting of buffer, ATP, CTP, CTP labeled on the alpha-phosphate group by P32, single-strand DNA template, and the gene four protein, or GP four primase helicase.
Incubate the mixture at room temperature for five minutes. After five minutes, use a pipettor to remove two microliters of the mixture, and mix it with two microliters of formamide dye buffer in one of the 1.5 milliliters tubes prepared earlier. This is the no reaction control.
Add two microliters of 0.1 molar magnesium chloride to the reaction mixture and immediately start a timer. At 10 second intervals, manually withdraw two microliter samples of the reaction mixture, and mix with the formamide dye in the 1.5 milliliter tubes. After all time points have been collected, heat the samples in a heat block at 95 degrees Celsius for five minutes.
Load aliquots of the samples onto a denaturing gel for electrophoresis. Begin this procedure by preparing the reaction mixtures. Prepare 400 microliters of solution A containing buffer, GP four hexamer, single-strand DNA template, dNTPs, ATP, CTP, and CTP labeled on the alpha-phosphate group by P32.
Prepare 400 microliters of solution B, containing one X buffer and 20 millimolar of magnesium chloride. Next, set up the rapid quench flow instrument. Turn on the instrument, and after the main menu appears, type seven on the instrument keyboard to move the syringe driver to the home position.
Open the buffer syringe position to load. Fill syringe C with quench solution, then, using a 10 milliliter syringe, fill the instrument buffer reservoirs A and B with buffer. Remove air bubbles by pushing plungers in and out.
Change the knob positions to fire. Type two to adjust the position of the syringe driver bar. Press the adjust down button to lower the syringe until the buffer comes out the exit loop.
Type escape to return to the main menu. To wash the tubing, attach the exit line to a vacuum. Change the sample knobs to flush.
Set the knobs to the horizontal position to close the buffer syringe. Turn on the vacuum, dip the two flush loops in water for 10 seconds, and then dip in methanol for 10 seconds. Dry the loops by sucking air for about 15 seconds, or until dry.
To load samples, change the sample knob position to load. Place solution A into a one milliliter syringe, plug the syringe into one of the sample load ports, in the same way, place solution B in the opposite sample load port. Begin the collection of time points by typing one on the main menu for a quench flow run.
Enter the reaction time and press enter. The reaction loop number to use will be displayed. Switch to the required reaction loop.
After washing the tube as demonstrated earlier, turn the sample knobs to load. Load the reaction mixes in the sample loops until just outside the central mixing compartment. Turn all the knobs to fire, and confirm that all knobs are in the correct position.
Place a 1.5 milliliter tube onto the end of the exit loop. Press go to run the reaction. After repeating the tubing wash procedure, reload buffer syringes A and B until they hit the buffer syringe driver.
Repeat the steps for running the reaction for each desired time point, with one exception, do not load solution B when quenching the zero time point control. When the sample collection is finished, enter escape to return to the main menu. Press seven to return the buffer syringe driver to its home position.
Start the instrument clean up by removing the reaction mixture syringes, turn the sample loading knob to the load position. Under vacuum, wash each sample loop with 10 milliliters each of sodium hydroxide, phosphoric acid, and methanol. Press down on all three syringe plungers to retrieve the reaction and quench buffers.
Wash the syringes with five milliliters of water at least twice. Fill the syringes again with five milliliters of water, turn the knobs to the fire position, turn on the vacuum and bring down the driver via the keyboard command to remove the water. Wash the tubing as demonstrated earlier.
Lastly, turn off the rapid quench flow instrument. The samples are subsequently analyzed as described in the text protocol. These results were obtained by manually sampling a primer synthesis reaction catalyzed by GP four under multiple turnover conditions.
The labeled precursor CTP shows the highest mobility and migrates towards the bottom of the gel, followed by slower migrating species, corresponding to the dye and tetraribonucleotides synthesized by GP four. Depending on the length of incubation and template sequence context, additional labeled products corresponding to higher oligomers can be observed. When a rapid quench instrument is used to examine the reaction catalyzed by GP four in timescales in the range of seconds down to a few ms, it is evident that primer accumulation is not linear, but displays a biphasic profile indicated by the solid line.
This suggests that product formation is rapid, and release is rate-limiting in the steady state. The dotted line represents a hypothetical progress curve that would result if primer formation by GP four did not proceed with a pre-steady state burst. A single-turnover primer synthesis experiment provides information about the formation and decay of intermediate species during the conversion of substrate to product.
A time course of tetramer formation is shown by the gel image. The graph shows intermediate formation versus time. Once mastered, this technique can be done in 90 minutes, if it's performed properly.
Don't forget that working with radioactive reagents can be extremely hazardous and precautions such as proper shielding should always be taken while performing this procedure.
