Lab_2,_DNA_damage_assay,_2024

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Feb 20, 2024

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Lab 2: Antioxidants & oxidative DNA damage What’s going on today? Lab 2: DNA damage assay Setup for lab 5: Parental fruit fly cross Extract plasmid DNA using a spin column ↓ Setup & run DNA damage assay ↓ Run DNA samples on agarose gel ↓ Assess antioxidant activity of your test compounds ↓ Complete in-class worksheet *Due at 11:59 pm today* ↓ Experiment mini-summary *Due at start of lab 3, two weeks from today* Anesthetize WT and mutant fruit fly cultures ↓ Observe sexual dimorphisms & mutant phenotypes ↓ Lab coordinator will set up crosses for you Lab 2 Introduction Oxidative DNA damage and reactive oxygen species (ROS) Oxidative damage to DNA and other cellular components is a major concern in various fields of study. Oxidative DNA damage can include modifications to the DNA base pairs or the DNA sugar-phosphate backbone. Oxidative DNA damage can lead to genetic mutations or cell necrosis. The effect of oxidative damage on cells and tissues is a major mechanism involved in aging and in the development of neurodegenerative diseases, cancer, or other pathologies. Oxidative damage is often caused by reactive oxygen species (ROS) and the free radicals they generate. ROS ’s are produced 1 There is a lot of well documented evidence around the harm caused by oxidative damage and the important role of antioxidants. However, claims in naturally by the body as a byproduct of normal cellular metabolism, but they can also be produced by external sources such as pollution, radiation, and UV light. Antioxidants are molecules that can neutralize free radicals and help protect DNA and other cellular components from oxidative damage. Cells naturally produce many proteins and small molecules with antioxidant properties, and antioxidants can also be obtained through the diet or by taking nutritional supplements 1 . Plasmid DNA molecules Plasmids are small circular DNA molecules that are contained within bacterial the media or non-scientific sources about the health benefits of eating “superfoods” or taking antioxidant supplements are highly dubious and should be viewed with a healthy dose of skepticism.

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 2 cells in addition to t he bacteria’s chromosomal DNA. The role of plasmid molecules in bacterial evolution or their use in molecular biology applications are both interesting topics, . For example, one of the mechanisms that bacteria use to rapidly evolve antibiotic resistance is by horizontal gene transfer via plasmid molecules. In simple terms, one cell evolves an antibiotic resistance gene that’s encoded on a plasmid molecule, then that cell can very quickly pass copies of the resistance plasmid to other cells. As another example, plasmids are routinely used in molecular biology or biotechnology research to customize genes and/or express these genes in other systems. One pertinent application is to use a DNA plasmid encoding the spike protein gene from the SARV-CoV2 virus to transcribe mRNA which is collected and used in COVID-19 vaccines. These examples are beyond the scope of this lab and details are best left for other courses ( e.g. microbiology, biotechnology, or molecular genetics). For this experiment’s purpose, you will use plasmid molecules as a convenient way to assess oxidative DNA damage . Isolating plasmid DNA from bacterial cells is an easy way to obtain relatively large amounts of DNA, while practicing some of the fundamental techniques used in a genetics lab. Plasmid DNA molecules can exist in a variety of conformations. The three most relevant conformations are supercoiled, nicked, and linear. Supercoiled plasmids are tightly wound up on themselves, giving them a compact overall shape (imagine an elastic band wrapped up on itself). Relatively mild DNA damage can lead to a single-stranded break in the plasmid DNA, which allows the supercoiled plasmid to unravel. This “nicked” conformation resembles an open loop (imagine the elastic band is now relaxed and uncoiled). More severe DNA damage can lead to a double-stranded DNA break. This would break the circular shape, leaving a linear DNA molecular. Plasmids are generally supercoiled when they are in the bacterial cells, but the extraction process tends to cause mild damage to some of the plasmid molecules. DNA damage assay & agarose gel electrophoresis The DNA damage assays you will run will involves incubating plasmid DNA with tin (ii) chloride alongside known or potential antioxidants. Tin 2 + ions generate ROS’s which could potentially damage the plasmid DNA. The assay conditions you’re using are sufficient to cause extensive damage to the plasmid DNA, unless an antioxidant protects it. After the assay has finished, you’ll examine the relative abundance of the three plasmid conformations – supercoiled, nicked, and linear – to determine how effectively the antioxidants were able to protect the DNA from oxidative damage. The conformation of plasmid molecules can be assessed using agarose gel electrophoresis . This is a technique that allows you to separate DNA molecules based on their physical properties – size, electrostatic charge, and shape. DNA generally has a uniform negative charge, so this property isn’t usually considered when electrophoresing DNA. In most cases where you would use gel electrophoresis for DNA molecules, they would have the same linear shape and their size would be the distinguishing factor. This experiment will be a rare case where all your DNA molecules will have the same size and will therefore be separated based on their shape . This separation is accomplished by loading samples into a porous gel matrix and applying an electrical current. The negative charge on the DNA backbone causes it to migrate in the direction of the current, towards the anode. The various conformations of plasmid DNA take on different 3D shapes, some of which migrate through the pores more quickly whereas other conformations are prone to getting stuck in the pores and migrating more slowly. The 3D shape of DNA

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 3 molecules is more complicated than often shown in textbook examples and is highly dependent on chemical conditions. For the sake of this worksheet, you can assume that the confirmations of plasmids resolve in the same order as is shown in figure 2.1 D. Experimental controls At the end of this experiment, you’ll look at DNA banding patterns on a gel and make conclusions about whether antioxidant molecules absorbed free radicals or not. There is a lot happening in your assays that you can’t see for yourself, and lots of assumptions you have to make . You assume that it’s true that SnCl 2 induces oxidative DNA damage and that antioxidants can prevent this damage. You’re assuming the various plasmid conformations will resolve on the gel as described above. You also assume that all the materials you were given were prepared correctly and that you performed the various techniques correctly. All of this is to say, that you should be skeptical of your results, and you need to convince yourself and others that the results you see actually support the conclusion you draw from them. An important way to convince yourself and others that your experiment is working as planned is to include experimental controls . Controls are samples where you already know what the results should be. When the control samples give the results you expect them to, you can feel confident to trust the results of your experimental (unknown) samples. If controls give different results than you expected, something must be wrong with your experimental setup or the assumptions you’ve made. You can’t trust the results of these experiments & need to refine your experiment and try again. For this experiment you’ll use two controls samples. One assay will contain no antioxidant at all and one will contain the known antioxidant ascorbic acid . Can you predict the level and DNA damage and banding pattern that you expect see on the gel for each of these controls?

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BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 4 ROS ROS Supercoiled Nicked Linear A) Figure 2.1: Principles of your DNA damage assay. A) Plasmid DNA begins in a supercoiled conformation and will become nicked by mild oxidative damage, or linear through severe damage. B) Electron micrographs of supercoiled and nicked plasmid DNA molecules. C & D) Separation of supercoiled, nicked, and linear plasmid molecules on agarose gels. A DNA ladder is included, as is standard practice, though it’s not important for this lab. The buffer used for running agarose gels affects the order that plasmid conformations resolve in. TAE buffer (C) is most commonly shown in textbooks etc , but we’ll use TBE buffer (D) in all of our labs. TAE buffer (Shown to match textbooks etc ) C) Supercoiled Nicked Linear Super- coiled Nicked B) Linear Nicked Supercoiled TBE buffer (Used in all our labs) D)

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 5 Protocols Today’s lab: Extract plasmid DNA with a spin column You will use a spin column kit to extra plasmid DNA from provided E coli cells. A spin column contains a filter that will bind DNA under certain conditions, and release the DNA under other conditions. Liquid is put into the spin column, then it’s centrifuged to pass the liquid through the filter. Perform 1 plasmid isolation per pair of students. Explanation Detailed protocol The E. coli culture provided will have millions of cells per ml, and each cell holds ~50 copies of the plasmid DNA molecule. First, the chemically complex media the cells were grown in must be replaced with a simple buffer. 1. Transfer 1.5ml of E coli culture into a microtube. 2. Centrifuge at max speed for 1min. Make sure to balance your sample against another tube. Use a microtube with 1.5ml of water as balance if there is an odd number of students. 3. Decant the liquid from your tube into the liquid waste container. Remove residual liquid by sucking it up with a pipette tip or tapping the mouth of your open tube against paper towel laid on the bench. Don’t worry if a small drop remains. 4. Pipette 200μl of resuspension buffer onto your cell pellet. Pipette up and down several times to thoroughly mix the cells into suspension. The cell lysis buffer uses SDS (a detergent) & an alkaline pH to lyse (break open) the bacterial cells. RNAse A in the resuspension buffer quickly degrades the bacterial RNA. The neutralization buffer neutralizes the pH and potassium ions react with the SDS to form a white precipitate. Cellular proteins and chromosomal DNA precipitate along with the SDS. The plasmid DNA remains soluble in the supernatant. The high salt level of the sample at this point causes the plasmid DNA to adhere to the spin column filter. 5. Add 200ul of cell lysis buffer. Close the tube and invert 5-10x to gently mix. Let the sample sit for 2-5min for the cells to lyse. 6. Add 300ul of neutralization buffer and invert the tube 5-10x to mix. 7. Centrifuge at max speed for 5min. Again, balance against another sample or against 700μl of water. 8. Use a pipette to transfer the liquid into a spin column (which is itself inside a collection tube). Try to avoid the solid material – it’s better to leave a bit of liquid behind to make sure you don’t get any of the solids . Discard the microtube containing the solid material

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 6 Centrifuge the spin column assembly to pass the buffer through the filter. The DNA sticks to the filter, while most other material passes into the collection tube to be discarded. 9. Spin your column in a microcentrifuge for 30sec-1min. 10. Remove the spin column, dump the flow through into liquid waste, and replace the spin column into the collection tube. The DNA stays adsorbed to the filter, while any remaining materials are washed away. 11. Add 600μl of wash buffer to your column, and spin again for 30sec-1min. Put the lid back on the bottle of wash buffer quickly- don’t leave it open longer than necessary . 12. Discard the flow through, return the spin column to the collection tube, and spin for another 3min to dry the filter. Distilled water releases the DNA from the filter (due to lack of salt). After spinning you will have highly purified plasmid DNA in the microtube. 13. Transfer your spin column to a microtube labelled with your name or initials. Add 100μl dH 2 O onto the spin column. Let the column sit for 1-2min to allow the DNA to elute from the filter. 14. Spin your column inside the microtube (not the collection tube) for 1min to elute your purified DNA. The lid of the microtube will be left open for this spin. 15. Your purified DNA is in now in the dH 2 O in your microtube. Discard the spin column and collection tube. Today’s lab: DNA damage assay Prepare a set of 4 reactions per pair of students . You’ll do two control samples (one containing no antioxidant and one containing the known antioxidant ascorbic acid), and two unknown/experimental samples (prepared in the previous step). Note : Pipetting small volumes can be tricky! Before you expel liquid from your pipette, make sure the tip is either touching the inner wall of the tube wall or is inside a drop liquid that’s previously been added. This will ensure the small drop of reagent doesn’t just stick to the outside of your pipette tip after you expel. 1. Obtain a strip of 4 strip-tubes. Add 10 μ l of dH 2 O to each tube, then add 5 μ l of plasmid DNA to each tube. Finally, add 5 μ l of an antioxidant sample to each tube: − Tube 1: dH 2 O (negative control) − Tube 2: ascorbic acid (positive control) − Tube 3: Polyphenolate A (PPA, an experimental sample) − Tube 4: Polyphenolate B (PPB, an experimental sample) − 2. If needed, have a TA help you spin down your tubes to collect the liquid at the bottom.

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BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 7 3. Add 5 μ l of SnCl 2 to each of your four tubes. Try to do this quickly, so that all DNA is exposed to the SnCl 2 for the same amount of time. Try to keep the liquid at the bottom of your tube – spin down the tubes if needed. 4. Let the reactions sit for on your bench for 30min. 5. Stop the reaction by adding 5 μ l of EDTA to each tube. Again, do this quickly so that all reactions run for the same amount of time. Today’s lab: Preparing an agarose gel Prepare 1 gel for a group of 4 1. Ensure gel caster has a casting tray, and a comb with the small teeth facing down. 2. Weight 0.15g agarose and add it to a small flask. 3. Measure 15ml of TBE buffer and pour into the flask. 4. Microwave to dissolve the agarose, then let the flask cool until medium-warm 5. Pipette 5μl fluorescent dye into molten gel. Swirl to mix, then pour into one side of the gel caster. Wait 5-10min for gel to solidify. Today’s lab: Running and imaging your agarose gel Each gel will be shared by up to 4 students (2 sets of reactions). Each gel has 9 lanes - both pairs can load their 4 reactions and use the final lane to load a ladder. 1. Use a p20 to add 5μl of DNA loading dye into each tube. Mix by pipetting or flicking. 2. Load 15 μl of each reaction into separate lanes of the gel. You can adjust the light settings to your preference (high, low, off). 3. Load 10μl of DNA ladder into the final lane. 4. Place the lid on the gel tank. If the light next to the power button turns solid green, the gel is running. If the light is off, push the power button to turn it on. If the light is flashing, ask a TA to add more buffer to the tank. 5. Observe your gel periodically and use your phone to take a picture when the bands have adequately resolved. Using a foil covered gel tank lid will help take a brighter picture.

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 8 Setup for lab 5: Anesthetize and observe fruit flies ( Drosophila melanogaster) Note: The FlyNap solution you will use it non-toxic to humans, but it has a strong unpleasant smell. The FlyNap bottles should not be removed from the fume hood, and anaesthetization wands should be placed into the collection jars quickly once they are removed from the fly vials. Minimize the amount of time the wands are in the open air. Anyone who needs to step out of the lab for fresh air can do so without asking permission. 1. Collect two fly vials if you don’t already have them. The vials will either contain wild type flies (WT) or files with the mutant phenotype ‘ w m f ’ . Bring these vials to the fume hood. 2. Loosen the plug so that it is only a few millimeters into the vial. Dip an anesthetization wand in the FlyNap solution and dab off any excess on the lip of the bottle. 3. Tap the vial sharply on the counter to knock the flies to the bottom of the vial, then quickly squish the foam with your thumb to make a small opening. Insert the anesthetization wand into the opening, then release your thumb. Push the foam plug a bit deeper into the vial (just enough so it won’t fall out). 4. Repeat steps 2-3 with your second vial of flies. 5. Leave your vials in the rack in the fume hood and watch for them to stop moving. This should take 2-3 minutes from the time the FlyNap was introduced. 6. Once the flies fall to the bottom of the vial, remove the plug and quickly place the anesthetization wand in the collection jar and bring them back to your bench. . Remove the anaesthetization wands once the flies stop moving, even if you are not ready to work with them yet , or you risk killing the flies. 7. Tap the wild-type and mutant flies into opposite sides of a two-compartment petri dish, and observe them under a dissecting microscope, using the paint brushes to move or reposition the flies. See figure 2.2 for differences between males and females. Tip: Many students tend to set the magnification quite high. A lower magnification makes it easier to focus and to see multiple flies at once. 8. Identify three phenotypic differences between the WT and your mutant strains. Tip: The strain name ‘w m f’ reflects the three phenotypic difference of these flies compared to the wild type. The ‘w’ phenotype is very obvious and you can probably guess what ‘w’ stands for right away . The ‘m’ phenotype is more subtle , and you may or may not be able to guess what ‘m’ stands for. Identifying the ‘f’ phenotype will require close inspection of the flies and it’s unlikely you’ll be able to guess what the ‘ f ’ stands for (though you’ll still be able to see the trait if you look closely!). Note: Your lab coordinator will set up crosses of mutant females & WT males. You will observe the F1 offspring next lab.

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 9 Clean up instructions • Arrange all materials neatly on the bench & in the storage baskets (pipettes, tips & tubes, reagents, etc ). • Turn off, unplug and place the dust cover on dissecting microscope, set them to one side of the bench. • Make sure that lids of reagent bottles are all closed. • Discard o Spin columns & collection tubes from plasmid isolation o Left over plasmid (in 1.5ml microtube) o Strip tubes with left over DNA damage assays o Used tips & paper towel o Dump your small waste into the autoclave bins (BUT KEEP THE RED BAG) • Do NOT discard (leave in place for later groups) o DNA ladder, fluorescence dye for making gels, DNA loading dye o Plastic tube for measuring TBE buffer o Petri dished used for observing flies • Return any fly vials that smell like FlyNap to the fume hood. • Make sure your station has 2 lab copies of the protocols. • Tuck stools under bench. Figure 2.2: Anatomical differences between male and female Drosophila melanogaster . Note that the main differences are in the abdomen .

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BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 10 In-class worksheet [20 marks total] Results of a DNA damage assay similar to today’s lab is shown below. Consult this gel to answer questions 1 - 3. Refer to figure 2.1 panel D for a reminder of the order that plasmid conformations resolve in. 1. The first 5 lanes contain the following samples, but in a different order. Match the lane on the gel to the samples below. [5 marks] *** Options will be randomized on Brightspace 2 *** a. A control sample with a known antioxidant (very high antioxidant activity). b. A control sample with no antioxidant c. A control sample with no plasmid DNA d. An experimental sample showing low antioxidant activity e. An experimental sample showing high antioxidant activity 2. The next 3 lanes contain three different experimental samples showing various levels of antioxidant activity. Two samples show results that could be expected in this experiment, but one sample gives a banding pattern that should be impossible. [2 marks] a. Which lane shows the impossible banding pattern? b. Of the two possible samples, which shows higher antioxidant activity than the other? 3. Explain briefly ( 1-2 sentences ) how you arrived at your answer for question 2 part a. [2 marks] 4. All of the below statements are true. Which statement best represents a conclusion that would be drawn from today’s experiment? [1 mark] a. Supercoiled plasmids form less sharp bands than linear plasmids an agarose gel. b. Sn(II)Cl 2 generates ROS’s that cause oxidative damage to DNA. c. The plasmid isolation procedure can be completed within 1hr. d. The experimental test compounds display varying levels of antioxidant activity. e. Samples need to be balanced before centrifuging. 2 Sorry, we can’t turn this off.

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 11 5. Before you could perform today’s lab, optimal assay conditions needed to be determined through trial-and-error. Below are 4 experiment trials with sub-optimal conditions. Each trial contains the same samples as the gel from question 1. Match each trial with the conclusion you would have drawn and how you would try again to find optimal conditions. [4 marks] Hard mode: Try this problem without looking at the possible options Regular mode: Look at the list of possible answers at the end of this document For today’s lab, you were provided with a solution of 10mM Sn(II)Cl 2 , which would have been diluted from a 200mM stock solution. 6. How would you prepare 2.5ml of 10mM Sn(II)Cl 2 starting from the 200mM stock solution and dH 2 O? Mix ___ ml of stock solution with ___ ml dH 2 O [1 mark] 7. How many grams of Sn(II)Cl 2 would have been needed to prepare 1mL of the 200mM Sn(II)Cl 2 stock solution? (Molar mass = 198 g/mol ; give your answer to 4 decimal places) [1 mark] 8. Weighing such a small amount is difficult to do accurately. It is better to weight an amount close to this, and adjust the volume of solvent you dissolve it in. If you were to weight out 54.2mg of Sn(II)Cl 2 , how many ml of water would you dissolve this in to get a 200mM solution? Give your answer to 3 decimal places. [1 mark]

BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 12 9. In later labs you will use gel electrophoresis with DNA produced by the polymerase chain reaction (PCR). DNA made by PCR is always linear and is generally much shorter than plasmid DNA. (FYI - The plasmid in today’s lab is ~7 kilobases (kb) long, whereas PCR products in later labs will all be less than 1kb). We will make and run the gels using the same equipment as today’s labs, but the gels will be made differently because of the size difference of the DNA molecules to be resolved. How do you think we’ll modify the gels when we deal with shorter DNA fragment s? ( Answer in 1 sentence ). [2 marks] 10. What biological source is agarose isolated from? [1 mark]

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BIOL 2104, Lab 2: Antioxidants & oxidative DNA damage 13 Possible answers for question 5 *** Options will be randomized on Brightspace *** a. Too much DNA was used – use less next time b. Too little DNA was used – use more next time c. The SnCl 2 was too concentrated – dilute it further next time d. The assay was not run long enough – incubate for longer next time

Lab_2,_DNA_damage_assay,_2024

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