The overall goal of this procedure is to analyze DNA polymerase activity on a single nucleotide level. This method can help answer key questions in the DNA polymerase field such as which specific steps of DNA synthesis are limited by DNA modifications. The main advantage of this technique is that you can observe single nucleotide changes enabling precise observation of polymerase activity.
While attempting this procedure it is important to remember to plan ahead. Many of the steps are time-sensitive. To begin this procedure first prepare the buffers, DNA duplex mix and protein dilution as outlined in the text protocol.
Next, set the temperature of a dry bath to 50 degrees Celsius. Add 24 microliters of the annealed duplex mix to a microcentrifuge tube containing 25 microliters of 2X dNTPs. After this, transfer 9.8 microliters of this mixture to a microcentrifuge tube containing 20 microliters of quenching buffer orange to prepare the no enzyme control.
Then add 0.8 microliters of 50X enzyme to the duplexed dNTP mixture. Use a large volume pipette to thoroughly mix the solution. Incubate in the dry bath.
Quench the reaction by removing 10 microliters of each reaction mixture and then adding it to a microcentrifuge tube containing 20 microliters of quenching buffer orange. Prepare the acrylamide mix for gel electrophoresis as outlined in the text protocol. Next, wash two 33-centimeter by 42-centimeter gel plates thoroughly with soap and water.
Dry each plate face with paper towels and then use delicate task wipers to dry the remaining areas. Rinse the dried plates with 70%ethanol and wipe each again with task wipers. After this, run a gloved hand under deionized water and then using wetted fingers gently wet some 0.75 millimeter spacers.
Place the spacers along the edge of the notched plate. Use task wipers to clean up any water that may have splashed on the plates. Put the plates together and then use gel tape to cover the bottom and sides leaving the top untaped.
Make sure the tape is aligned evenly and sealed tightly. Using a razor cut the ends of the tape. Then add an additional layer of tape to the bottom of the gel.
After preparing the reagents, acquire a squirt bottle and remove the pointed funnel. Using a graduated cylinder, add 125 milliliters of water. Then use a permanent marker to mark the level of the water on the outside of the bottle.
Discard the water. Next set up the cork rings and plate sandwich next to a sink as outlined in the text protocol. Place the empty squirt bottle and the prepared APS and TEMED solutions on the other side of the sink.
Place a well comb with the desired number of wells and four clamps nearby. Then add 20%acrylamide gel solution to the squirt bottle until it is at the marked level. Simultaneously add 120 microliters of TEMED and 600 microliters of APS to the polyacrylamide gel solution.
Quickly add an additional 600 microliters of APS. Swiftly cap the squirt bottle and mix by swirling. Next place the plate sandwich into the sink.
Begin pouring, applying slow and steady pressure to the bottle to ensure the gel solution flows steadily. Pour until the gel has reached the notched area and is slightly overflowing from the notched edge. Tilt the plates to remove any air bubbles.
Then add the well comb to the plate sandwich. Place the clamps on the comb such that they press down allowing for even distribution of the wells. Quickly dispose of any remaining gel solution in the squirt bottle to an appropriate waste container.
Rinse with the deionized water three times through the squirt nozzle to clean the bottle. To begin remove all binder clips from the plate sandwich. Using a razor, cut and remove the gel tape from the edges.
Wipe the glass surfaces with paper towels and water to clean off as much polymerized gel residue as possible. Use a razor along the edges to scrape off any remaining gel. Next push the well comb evenly on both sides to remove it.
Using a razor, cut off any excess gel along the top of the plate sandwich. Then slice along the notched edge of the plate. Using a syringe and deionized water, rinse the wells to remove any debris.
Make sure that each well can be filled with water and is not blocked by excess polymerized gel. Next place the plate sandwich into the apparatus so that the longer plate is facing outward while the notched space is facing inward. Clip the plate sandwich and aluminum plate together attached to the apparatus.
Add additional clips to the outside of the plate to promote even running. Add TBE buffer using just enough to cover the base notches and the wells at the top. Run the gel electrophoresis at 50 watts for 20 minutes to warm the plates.
After this use a syringe and TBE buffer to clean the wells. Repeat the syringe wash several times to ensure that all remaining buffer salts have been removed from the wells. Next set up the outermost lanes on each side as outlined in the text protocol.
Add three microliters of 95%formamide with bromophenol blue to the appropriate wells of the gel. Load all samples and allow them to settle for one minute. After connecting the apparatus and power source, set the power to between 50 and 55 watts.
Run the gel for approximately three hours or until the bromophenol blue dye front has run at least two-thirds of the way down the gel. After this, image the gel as outlined in the text protocol. In this study an assay is developed to characterize the DNA polymerase-mediated synthesis of modified DNA.
A successful qualitative characterization of overall activity shows individual bands that are well-defined and regular. Activity is judged by both the length of the products and the portion of the labeled primer that is converted to larger products. As can be seen, E6 and E5 are the most active enzymes, while E4 and E1 are the least active.
A quantitative analysis using steady-state kinetics can also be performed using these methods. Because only the N and the N+1 products are synthesized, the singular event can be quantified successfully. The basic methodology here can also be used to study a number of other DNA or RNA modifying enzymes other than polymerases.
