20.109(F16):Measure DNA damage with comet chip (Day3)

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20.109(F16): Laboratory Fundamentals of Biological Engineering

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       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Introduction

In the initial experiment for this module, your goal was to optimize the CometChip assay. Now that you have data concerning how to best load cells into the CometChip, you will use this technique to study the Base Excision Repair (BER) pathway. BER corrects DNA damage, specifically base lesions, that result from oxidation, alkylation, deamination, and depuriniation/depryrimidination. If base lesions are not repaired, non-canonical base pairing can occur, which may result in the incorporation of an incorrect base during replication. To prevent mutations and maintain integrity of the genome, the BER pathway evolved as a highly conserved repair mechanism in both E. coli and mammals.

Base excision repair pathway.
The core BER pathway includes only four proteins that function to remove the damaged base and replace it with the correct base. First, a DNA glycosylase recognizes that a damaged base is present in the DNA and cleaves an N-glycosidic bond, which creates an apurinic or apyrimidinic site (referred to as an AP site in both cases). Different DNA glycosylases recognize different types of base lesions. Second, the DNA backbone is cleaved to create a single-strand DNA nick by either a DNA AP endonuclease or a DNA AP lyase. Next, a DNA polymerase incorporates the correct base using the sister strand as a template. Last, a DNA ligase completes the repair by sealing the single-strand nick, which restores integrity to the helix. For a more detailed description of the BER pathway, read this review by Robertson et al.

The BER pathway repairs base lesions using either a short-patch or long-patch mechanism. In the short-patch BER pathway, a single nucleotide is removed in the repair process. In contrast, in the long-patch BER pathway at least two nucleotides are removed. Though both mechanisms of repair are utilized, the conditions that drive each are poorly understood.

To study the BER pathway, as a class you will use two chemical treatments to generate base lesions. The treatment will be applied to both live cells and dead cells. The use of dead cells provides an interesting control because the researcher can interrogate the precise level of damage induced by a treatment when the repair mechanisms are not active in a cell.

Protocols

Part 1: Examine CometChip loading experiments

In a group discussion with the teaching faculty, you will assess the results of the class data from the CometChip loading experiments. The goal here is to determine which cell number and loading time to use when preparing your CometChip for the tests below.

Be sure to include notes on the discussion and the values for cell number and loading time that you will use in your notebook!

Part 2: Induce DNA damage

You will load the CometChips you prepared on M1D1 using the procedure from M1D2; however, please note the following differences:

  • The procedure for 'trapping' the cells into the microwells will be as follows:
  1. Retrieve one tube of molten 1% low melting point (LMP) agarose from the front laboratory bench.
    • You will need to work quickly from this point as the 1% LMP agarose will solidify as it cools.
  2. Using the P1000, pipet up 1000 μL.
  3. Hold the pipet tip over the top left well of your CometChip and as you expel the agarose move the pipet tip from left to right. Complete this process for every row of your CometChip.
    • The goal is to lightly cover the wells that contain cells, which will 'trap' the cells into the microwells.
  4. Leave your CometChip undisturbed on your bench for 3 min, then move it to the 4 °C cooler for 3 min.
  • For the cell number and loading time, you will use the values decided on during the group discussion in Part 1.

After you load the cells into the CometChip, you will induce DNA damage using either H2O2 or MMS as assigned by your teaching faculty. Follow the appropriate protocol for damage induction based on the chemical you were assigned to test.

DNA damage inducer: H2O2
In addition to the CometChip you loaded above, you will also treat a CometChip that contains dead cells. The CometChip with dead cells was prepared by the teaching faculty and should be retrieved from the front laboratory bench. To be clear, you will complete all of the below steps with the CometChip you loaded, which will contain live cells, and the CometChip that contains dead cells.

  1. While the LMP agar is solidifying at 4 °C, calculate the dilutions of H2O2 that you will use for your experiment.
    • The H2O2 stock solution is at a ~10 M concentration.
    • You will add 100 μL per well to induce DNA damage.
    • Calculate dilutions that result in doses of 0, 10, 20, 40, 60, and 80 μM.
  2. Retrieve your CometChip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
    • Secure the bottomless 96-well plate with binder clips as before.
  3. Obtain an aliquot of H2O2 and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
    • Be sure to keep the H2O2 on ice at all times!
  4. Using a multi-channel pipet, add the appropriate treatment doses of H2O2 to each well of your CometChip.
    • The 0 μM treatment should be added to the wells at the far left and the 80 μM treatment should be added to the wells at the far right of the CometChip.
  5. Carefully transport your CometChip to the 4 °C cooler and incubate for exactly 20 min.
  6. Retrieve your CometChip from the 4 °C cooler and quickly remove the bottomless 96-well plate while holding the CometChip at an angle such that the low doses of H2O2 'run' over the wells with high doses.
    • If the high doses of H2O2 run over the low dose wells, additional damage may be induced and confound the results.
  7. Rinse your CometChip using 1x PBS as done during the cell loading procedure.
    • Complete a total of 3 washes.

DNA damage inducer: MMS
In addition to the CometChip you loaded above, you will also treat a CometChip that contains dead cells. The CometChip with dead cells was prepared by the teaching faculty and should be retrieved from the front laboratory bench. To be clear, you will complete all of the below steps with the CometChip you loaded, which will contain live cells, and the CometChip that contains dead cells.

Please be very careful when using MMS as it is a mutagen. You should always wear flock-lined gloves, in addition to the standard laboratory gloves, when using MMS.

  1. While the LMP agar is solidifying at 4 °C, calculate the dilutions of MMS that you will use for your experiment.
    • The MMS stock solution is at a ~12 M concentration.
    • You will add 100 μL per well to induce DNA damage.
    • Calculate dilutions that result in doses of 0, 0.25, 0.5, 1, 2, and 4 mM.
  2. Retrieve your CometChip from the 4 °C and carefully replace the bottomless 96-well plate to recreate the wells that you used for cell loading.
    • Secure the bottomless 96-well plate with binder clips as before.
  3. Obtain an aliquot of MMS and prepare the doses that you calculated in Step #1 using 1x PBS as the diluent in a 12-well reservoir as discussed in prelab.
    • Work on a piece of diaper paper at your bench when preparing MMS solutions!
  4. Using a multi-channel pipet, add the appropriate treatment doses of MMS to each well of your CometChip.
    • The 0 mM treatment should be added to the wells at the far left and the 8 mM treatment should be added to the wells at the far right of the CometChip.
  5. Carefully transport your CometChip to the 37 °C incubator for exactly 30 min.
  6. Retrieve your CometChip from the 37 °C incubator and quickly remove the bottomless 96-well plate while holding the CometChip at an angle such that the low doses of MMS 'run' over the wells with high doses.
    • If the high doses of MMS run over the low dose wells, additional damage may be induced and confound the results.
  7. Rinse your CometChip using 1x PBS as done during the cell loading procedure.
    • Complete a total of 3 washes.

Part 3: Complete CometChip assay

  1. Obtain a dish from the front laboratory bench and an aliquot of alkaline lysis solution.
  2. Place your CometChip in the dish and add the alkaline lysis solution.
    • Be sure the CometChip is completely submerged and not floating.
  3. Move the dish with your CometChip to the 4 °C cooler and incubate for 1 h.
  4. Remove your CometChip from the lysis solution and use a kimwipe to dry the gelbond side.
  5. Carefully transport your CometChip to the gel electrophoresis station in the 4 °C cold room.
    • The teaching faculty will escort you.
  6. Place your CometChip on the raised center region of an electrophoresis box.
    • Double-sided tape was applied to the gel electrophoresis box. Be sure you lay your CometChip on the tape strips and lightly press down with a pipet tip to ensure it is secure.
  7. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
  8. Your CometChip will incubate in the electrophoresis buffer for 40 min to promote unwinding of the DNA.
  9. To separate the damaged DNA into 'comets' it is important that the electrophoresis occur at 300 mA. To maintain the appropriate current, the volume of electrophoresis buffer may need to be adjusted. The teaching faculty will assist you in adding/removing electrophoresis buffer such that this value is reached.
  10. The electrophoresis will continue for 30 min (at 16 V, or 1 V/cm).
  11. Carefully remove your CometChip from the electrophoresis box and place it in a dish.
  12. Obtain an aliquot of neutralization buffer from the front laboratory bench.
  13. Wash your CometChip by adding enough neutralization buffer to cover and incubate for 5 min at room temperature.
    • Repeat this step a total of 3 washes.
  14. Add the SYBR gold DNA stain to your CometChip and carefully move it to the 4 °C cooler.

The teaching faculty will image your CometChip after ~16 hr and provide the images to you in the next laboratory section.

Reagents list

CometChip:

  • agar, normal melting point (Invitrogen)
  • phosphate buffered saline
  • GelBond film (Lonza)
  • 1 well dish (VWR)
  • agar, low melting point (Invitrogen)
  • bottomless 96-well plates (VWR)

Cell culture:

  • TK6 cells (ATTC CRL-8015)
  • RPMI 1640 (Invitrogen)
    • with 10% fetal bovine serum
    • and 1% penicillin/streptomycin
  • Trypan Blue
  • MMS: methyl methanesulfonate (Sigma Aldrich)
  • H2O2: hydrogen peroxide (Sigma Aldrich)

Alkaline lysis solution: (Sigma Aldrich)

  • 2.5 M NaCl
  • 100 mM Na2EDTA
  • 10 mM Tris
  • pH 10

Alkaline electrophoresis solution: (Sigma Aldrich)

  • 0.3 MNaOH
  • 1 mM Na2EDTA
  • pH 13.5

Neutralization buffer: (Sigma Aldrich)

  • 0.4 M Tris
  • pH 7.5

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