The overall goal of this trans-Pacific primer extension QPCR essay, is to quantitatively assess the repair of adducts, cross-linked to plasmid DNA, following transfection into mammalian cells. This method could help answer key questions in the DNA repair field. Such as what pathways are involved in the repair of DNA protein crosslinks.
This technique has the potential to provide a better understanding of the DNA damage response. Because it permits one to selectively study the repair of DNA protein crosslinks and the absence of other types of DNA damage. Though this method can provide insight into the repair of DNA protein crosslinks, it can also be applied to other types of DNA damage.
Such as abasic sites, and other polymerase blocking adducts. The main advantage of this technique, is that it can detect repair of adduct-containing plasmids, at time points as early as two hours. We initially intended to use house cell reactivation to study repair however those assays don't directly measure repair or may overestimate repair efficiency.
Particularly if RNA polymerase can read through incomplete repair products. Visual demonstration of this method is critical as the purification and strand-specific primer extension steps are otherwise difficult to visualize. Mix 20 microliters of a solution containing 80 picomoles of an eight oxoguanine containing oligonucleotide with five microliters of 10x ligase buffer.
And add one microliter of a solution containing 10 units of T4 polynucleotide kinase. Adjust the final volume to 50 microliters and incubate the tube in a water bath for 30 minutes at 37 degrees celsius. Next constitute the primer-extension reaction on ice.
Set the program on the thermo-cycler and start the run. After incubation, adjust the total volume to 375 microliters by adding different reagents, enzymes and buffer. Then incubate the reaction in a water bath at 37 degrees celsius over night.
To prepare a phenyl-chloroform mixture at a ratio of 50:50, add equal volumes of phenol and chloroform. Then mix both the components and spin in a table-top centrifuge at 21, 130 G for five minutes. After the spin is over, add 375 microliters of the organic layer to the primer-extension reaction.Mix.
And again centrifuge at 21, 130 G for five minutes. After centrifugation, carefully pipette the top layer. And mix with ammonium acetate to a final concentration of 0.3 molar.
And then add two volumes of 100%ethanol to it. Store the mixture at negative 20 degrees celsius for at least 30 minutes to overnight. Once the incubation period is over, centrifuge the sample at 15, 000 RPM at four degrees celsius for 10 minutes.
Then discard the supernatant and wash the pellet in 70%ethanol. Spin the sample again in a table-top centrifuge at 15, 000 RPM for five minutes at four degrees celsius. After the centrifugation, discard the supernatant and dissolve the pellet in 100 microliters of water.
Combine 50 microliters of the DNA with 34 microliters of water and 16 microliters of 6x gel-loading dye. Run the sample on a 10 centimeter, 0.8%low-melt agarose gel containing 0.5 micrograms per milliliter of ethidium bromide at two volts per centimeter for six hours in 1x TAE buffer. Then use a razor blade to excise the supercoiled DNA.
And weigh the gel slice. Next add one microliter beta-agarase reaction buffer to digest every 10 milligrams of gel slice. Then incubate the slice at 65 degrees celsius for 10 minutes followed by cooling at 42 degrees celsius in a thermo-cycler.
Once the gel slice has dissolved and cooled down to 42 degrees celsius, add 10 units of beta-agarase and leave at 42 degrees celsius for one hour in the thermo cycler. After one hour measure the volume of the dissolved gel slice and add ammonium acetate to a final concentration of 0.3 molar and incubate on ice for 15 minutes. After incubation, centrifuge the mixture at 15, 000 G for 15 minutes at room temperature.
Then collect the super natant and pipette two volumes of isopropanol to it and mix. Then store the mixture at negative 20 degrees celsius over night. The following day, centrifuge the purified supercoiled DNA in a table-top centrifuge at 15, 000 RPM for 10 minutes at four degrees celsius.
Remove the super natant and resuspend the pellet in 40 microliters of water. Then combine 15 microliters of a solution containing 12 picomoles of DNA with one microliter of a solution of 36 picomoles of oxoguanine glycosylase in a buffer and adjust the final volume to 30 microliters. Then incubate the mixture at 37 degrees celsius for 30 minutes in a water bath.
A day before the transfection, seed the cells in a six-well culture plate. The following day mix 1.5 micrograms of the cross-linked plasmid with 300 microliters of serum-free culture medium in one tube, making sure to save one microliter of DNA as a zero-hour sign point. In another tube mix 12 microliters of transfection reagent with 300 microliters of serum-free medium.
Then combine 300 microliters of the cross-linked DNA with an equal volume of diluted transfection reagent. And incubate for five minutes at room temperature in a laminar flow hood. After the incubation, add 250 microliters of the complex to each of the two wells and incubate at 37 degrees celsius.
After a minimum of one hour, remove the medium and add one milliliter of 0.6%sodium dodecyl sulfate solution with 0.1 molar EDTA and incubate at room temperature for 10 to 15 minutes. Then detach the cells by scraping with a rubber policeman and transfer to a 1.5 milliliter microfuge tube. Next add 200 microliters of 5 molar sodium chloride solution to a final concentration of one molar and invert the tube five times.
Then incubate at four degrees celsius overnight. The next day centrifuge the sample at 21, 130 G for 30 minutes at four degrees celsius. Post centrifugation, collect the super natant.
And add ammonium acetate to a final concentration of 0.3 molar, mix and add two volumes of 100%ethanol to precipitate the DNA. Store the mixture at negative 20 degrees celsius for a minimum of 30 minutes. Following ethanol precipitation and resuspension of the recovered DNA samples in 50 microliters of water, dilute the zero-hour sample in 500 microliters of water and constitute the PCR reactions using the non-transfected and transfected samples.
Then set the program on the thermo cycler and start the run. This trans-specific primer extension step is crucial because it buys us amplification of the damaged strand. If not used, delta CT values would be less than one, making it difficult to detect low levels of DPC repair.
After completing eight cycles, add 100 picomoles of the second primer. Then mix one microliter of the unamplified DNA from the non-transfected and transfected samples with 2x master mix, water and 100 picomoles of both primers to a final volume of 60 microliters. Load the samples in triplicates on a 96-well PCR plate.
Perform quantitative PCR for 30 cycles and average the cycle threshold for each of the triplicate samples. In this study QPCR is performed with and without strand-specific primer extension in order to calculate the percentage of plasmid DNA that has been repaired. The difference in the cycle threshold values between the primer-extended and the non-primer-extended samples is referred to as delta CT.As seen here, a repaired sample subjected to SSPE-QPCR has a larger delta CT than an unrepaired sample.
Representative data of the percent repair calculated from delta CT values shows that samples recovered after three hours of transfection are 66%repaired while samples recovered after eight hours are 93%repaired. Percent background values calculated from two control samples with respectively high and low protein crosslinking efficiency are shown here. The low-percent background present in the control, indicates why only efficiently crosslinked substrates are used for transfections.
Once mastered this technique can be done in two to three hours. While attempting this procedure, it's important to take time zero samples to subtract out any background from this assay. Following this procedure, other methods like sequence analysis can be performed to answer additional questions like:is DPC repair error prone?
After watching this video you should have a good understanding of how to prepare, transfect and quantify repair of adduct-containing plasmids in mammalian cells. Don't forget that working with phenol, chloroform and ethidium bromide can be hazardous. And precautions such as wearing gloves and working in a fume hood should always be taken.
