Damage-sensing PCR (dsPCR)

Damage-sensing PCR (dsPCR)

Assay description and experimental design 

Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of DNA damage that prevents PCR amplification. Exposure to DNA-damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. The method successfully detects damages induced by ultraviolet (UVC) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene of interest in repair. Furthermore, the use of a bead-based protocol for DNA extraction makes this method fully amenable for high-throughput screening of DNA repair activity. 

The underlying premise of our dsPCR assay (Figure 1) is that damages will block replicative DNA polymerases and this will result in lower PCR signal. A single damage along the amplicon will prevent amplification of the downstream primer sequence, thus preventing successful exponential amplification by PCR. For efficient detection, the majority of DNA fragments must indeed contain damage within the amplified region. However, when repair is also tested, it is crucial to treat cells with sub-lethal damaging doses. Commonly used treatment doses result in genomic damage frequencies in the order of 1:5000 nucleotides (nt). Primers should be designed to amplify amplicons of ~10Kb that would assure the majority of fragments indeed contain damages. The amplification of these long templates requires two special considerations: first, the use of a highly processive DNA polymerase that is able to efficiently amplify the long template from human genomic DNA. Second, gentle DNA extraction that would avoid excessive genome fragmentation.

For each primer pair, PCR conditions are calibrated for: 1) optimal annealing temperature that would give a single product and 2) the number of PCR cycles that are below saturation, to obtain a linear increase in PCR product with the increase in template concentration. 

            To account for errors in initial DNA template amount, in parallel to the long PCR, it is important to perform PCR of a short ~200bp fragment. At the damage doses used, the effect of damages on the amplification of this short fragment are expected to be negligible. To verify reproducibility, perform experiments in biological replicates. A list of validated primers is provided in Table 1.

Materials and Reagents

Cisplatin [cis-diamminedichloroplatinum(II)] 1mg/mL stock solution, Pharmachemie BV

BPDE stock solution (5mM), MRIglobal, Cat.477. 

• Dissolve BPDE powder in appropriate DMSO solution to obtain 5mM concentration. Aliquot BPDE in 25mL volumes to avoid freeze-thaw. 

Phosphate buffered saline (PBS) without calcium chloride, Sigma Aldrich Cat. D8537-500ML

Dimethylsulfoxide solution (DMSO), Fisher Scientific Cat. BP231-100

Growth media used for your cell line of choice

Trypsin solution (0.25% Trypsin, 0.02% EDTA), Sigma Aldrich Cat.T4049

MagAttract HMW DNA purification kit, Qiagen Cat. 67563

QuantiFluor dsDNA system, Promega Cat. E2670

LongAmp Taq 2X Master Mix New England Biolabs Cat. M0287L

Molecular biology grade water (DNase RNase-free), Biological Industries, Cat. 01-869-1B

1Kb extended marker, NEB Cat. N3239S

2100 SuperLadder, Bio-Lab Cat. 9597580SL2100

6x gel loading dye, NEB Cat. B7024S

Equipment

UVC lamp 254 nm, UVP XX15S, 95-0042-09 ( or any UV crosslinker, for UV experiments only)

Qubit fluorometer, Invitrogen Cat. Q33216

PCR, Bio-Rad C1000 Touch Thermal Cycler Cat. 1851148SP/1851196SP or similar

Overview of experimental procedure

Before preforming a dsPCR repair experiment, we advise to conduct a dose-response experiment to determine the optimal dosage of the agent to be used. The dose may vary between cell lines and should be adjusted. We recommend using a dosage that lowers the PCR signal by at least ~50%, ensuring that there is sufficient damage but is not too high to enable cells to recover and repair it. Cell survival can be estimated using cell survival assays such as  CellTiter-Glo® Luminescent Cell Viability Assay (Promega) or the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay (1) 

Step-by-step protocol

DNA damage induction and repair incubation

1. Seed cells in 6cm culture dish 24h prior to damage-treatment. 
2. Grow cells up to ~70% confluence.
3. Remove culture medium by gentle aspiration.
4. Wash cells with ~1mL PBS.
5. Treat cells with the chosen damaging agent: 
   1. For UVC treatment: Place plates uncovered in a UV-cabinet or crosslinker and treat with the appropriate dose. 
   2. For cisplatin treatment: Dilute 3.3mM cisplatin stock solution into growth media to make the wanted concentration, and add the cisplatin-containing media to the plates. Incubate for 2h in 37°C to allow damage formation. Continue to harvesting cells (step 6) or replace media for repair incubation. 
   3. For BPDE treatment:  Dilute 5mM BPDE stock solution into growth media to make the wanted concentration, and add the BPDE-containing media to the plates. Incubate for 2h in 37°C to allow damage formation. Continue to harvesting cells (step 6) or replace media for repair incubation. 
6. Harvest cells for DNA extraction. 
   1. Trypsinize cells in 1mL Trypsin for 5min in 37°C
   2. Collect cells into 10mL of ice-cold media in 15mL tubes. 
   3. Centrifuge the tubes for 5 minutes, at 4°C and ~500rcf. 
   4. Carefully aspirate the supernatant, without disturbing the cell pellet.
   5. Add 1mL ice-cold PBS and resuspend the cells with pipettor.
   6. Transfer the cells to a 1.5mL Eppendorf tube.
   7. Centrifuge the tubes for 5 minutes, at 4°C and ~500rcf. 
   8. Carefully aspirate the supernatant, without disturbing the cell pellet.
   9. Repeat PBS wash (steps v-viii)

***possible break-point: store pellet at -20°C for up to 3 months***

    7. Resuspend the cells in 200μL of ice-cold PBS

DNA extraction

  8. Extract high-quality genomic DNA from the cell pellets using Qiagen MagAttract HMW kit according to the manufacturer’s protocol. 

Note: To ensure that the MagAttract Suspension beads are fully resuspended place them on a vortex for ~10min.

Note: We found that other DNA extraction protocols resulted in long PCR failure. 

***possible break-point: DNA can be stored at -20°C for several months. We recommend aliquoting it to avoid multiple freeze-thaws***

    9. Genomic DNA quantification and dilution.

        Quantify genomic DNA concentrations using Qubit fluorometer and the Promega QuantiFluor dsDNA system according to the               manufacturer’s protocol. 

Note: Other fluorometric DNA quantification systems can be used. 

   10. Dilute genomic DNA into 10ng/mL stock solution and store in 100mL aliquots at -20°C.

Note: Having all samples with the same concentration simplifies later PCR set up. 

Damage-sensing PCR reaction

  11. Perform long-amplicon (damage-sensitive) and short-amplicon (normalizing) PCRs using the NEB LongAmp Taq 2X Master Mix           (NEB-M0287L). Perform 25mL reactions with 0.4mM of each primer and 20-100ng template DNA. Cycling conditions were         

        dependent on the primer pair (see “Determining PCR cycling conditions”).

Critical: Make sure to include a no template control in each experiment to monitor for PCR contaminations. We recommend using ultra-pure DDW and filtered tips in PCR setup as long amplicon PCR is very sensitive to contaminations. 

***possible break-point: PCR product can be stored at 4°C for several days or at -20°C for several months ***

   12. Quantify PCR product concentrations using Qubit fluorometer and the Promega QuantiFluor dsDNA system according to the    

         manufacturer’s protocol. 

Note: Other fluorometric DNA quantification systems can be used. 

Data analysis

DNA concentrations measured by qubit fluorometer after PCR of the long fragments should be normalized to the DNA concentration measured for the matching short-fragment PCR. These values can then be used to calculate lesion frequency as -ln([DNA]_Damage/[DNA]_Control)/PCR fragment length. 

To estimate repair efficiency, calculate the ratio of PCR product after repair incubation to the initial time point ([DNA]_Repair/[DNA]_T0). 

Design and quality control of PCR primers for damage detection

Primers for large and short fragments (20-25nt, Table 1) are designed with Primer 3 (2), with %GC set to 40-70% and Tm set to 65-68°C (NEB LongAmp Taq extension temperature is 65°C and determines the upper limit for annealing). 

Initial PCR is performed to verify the designed primer pair produces a single DNA product of the expected length and to establish the ideal annealing temperature. A 30-cycle gradient PCR is generally performed with annealing temperatures ranging from 5°C below the Tm to 65°C. To verify only a single band of the expected length is produced, the PCR products (5-10µL) are mixed with loading dye and separated on a 0.8% agarose gel alongside the NEB 1Kb extended DNA marker. 

Determining PCR cycling conditions

Perform calibrations to define the number of PCR cycles that are below saturation and provide a linear increase in PCR product with the increase in template concentration. Set up five PCR reactions with increasing amounts of template DNA (0, 20, 40, 80, and 100ng) for each cycling condition. For long fragments, PCRs of 26-30 cycles will generally yield linear amplification and can be tested in parallel by pausing the PCR machine and removing tubes after the completion of the relevant cycle. 

Troubleshooting/technical notes. 

• The most sensitive step of the process is preparing the PCR reaction. Any contamination at that point will result in high DNA concentrations in the no-template control.
• PCR steps should be done with dedicated pipettes and filtered tips. When possible, a dedicated closed environment (biological hood or PCR cabinet) should be used as well. 
• Molecular biology grade water (DNase RNase free, “pure DDW”) should be used in diluting the DNA and preparing the PCR reaction. It is recommended to use a fresh aliquot for each. 

While DNA concentration measurements are used for quantification, gel electrophoresis is best for troubleshooting the protocol and checking for contamination. Contamination will result in a strong “smear” of products in the no-template control (Figure 2). 

References

1.         T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63 (1983).

2.         A. Untergasser, I. Cutcutache, T. Koressaar, J. Ye, B. C. Faircloth, M. Remm, S. G. Rozen, Primer3--new capabilities and interfaces. Nucleic Acids Res 40, e115 (2012).