Science Buddies: "Ask an Expert"

What are the most common way to measure DNA damage?

Hi there!

Every organism has DNA, so depending oh what you are looking to study, there may be some different options. From the sciencebuddies main search page, I typed in DNA Damage https://www.sciencebuddies.org/science- ... dna+damage.

Here I found a lot of different project ideas and some other great information discussing DNA damage. I think it might be a great starting point for you and your project.

What kinds of organisms are you looking to study? What are the key questions you are looking to answer?

Please keep us posted as to how it goes and of course if you have other questions.

Good luck!
Nikki

Thanks for your reply. The key question that I want to answer is that how much damage does a single X-ray radiations do to the DNA?

How much damage does a single x-ray do to DNA?

Hi,

There is no such thing as a 'single' X ray. Radiation is continuous as long as the radiation source (X-ray machine, UV lamp, etc.) is ON. Maybe what you are thinking of is the 'dose' which is determined by the energy of the radiation and the time of exposure.

I think you need to read some more about radiation in general and its effects on biological materials such as DNA. It is a complex subject but the wikipedia entries

https://en.wikipedia.org/wiki/DNA_repair
https://en.wikipedia.org/wiki/Mutagen

explain what happens pretty clearly. Are you planning a project about radiation effects on living organisms? Let us know more about why you are asking about DNA damage.

Sybee

Thank you for the reply. What I want to do for my science fair experiment is that I wanted to see if I did X-Ray once a year how much damage will it do to the DNA and also what if I did it 6 times year or 12 times a year? I wanted to do as this my project because I heard that X-Ray is not good for our body and some people get X-Ray done and don't even know what happens to it? Once again thank you for the reply

You are certainly welcome!

I wish I could give you a simple answer for how to do a project like this, but radiation damage to DNA depends on many different factors--dose and dose rate, time, distance from the source, type of tissue, the presence of certain drugs and how well the cells can repair the damage.

The subject of how much DNA damage occurs from multiple exposures to X-rays has been pretty thoroughly explored over the past fifty years. Cancer is a disease that is associated with DNA mutations that can be caused by radiation damage. The more X-ray exposure a person has over their lifetime, the greater the risk that some of the damage will produce a specific change in the DNA that can cause a cell to lose control and start to divide without stopping.

The X-ray equipment in use today is much more sensitive and efficient than that used fifty years ago. It is able to obtain images of the body with very low X-ray doses that have minimal risk.

The effects of X-rays on DNA can be studied using lots of different model organisms including bacteria, yeast, plants, nematode worms, insects, mice, and human cells. If you want to do an experiment with X-rays, read about this Scibuddies project on zapping yeast with X-rays: https://www.sciencebuddies.org/science- ... p030.shtml

This project even has instructions for constructing an X-ray machine: https://www.sciencebuddies.org/science- ... p083.shtml

One area of radiation research that is still being actively studied today is the use of various dietary supplements like acai berries and blueberries to protect against the damage from ionizing radiation like X-rays (https://en.wikipedia.org/wiki/Ionizing_radiation). You could do a project to test a number of different supplements or extracts to see if they have protective effects. Do you have access to a lab to do experiments in? That would make a project like this much easier. Do you have a calibrated X-ray machine that you can use to administer a specific X-ray dose? If so, then you just need to choose an organism like yeast, feed it some acai or blueberry or vitamin E or A or C, give it an X-ray dose and see if it survives better than yeast that did not get a protective agent.

I hope this has given you enough information to focus your ideas better on this project. Let us know exactly what you want to do and we can help you with the details.

Good luck!

Sybee

Thank you for the reply. While I was reading the response from you, I was just wondering if the acai and blue berries are the protective agent?

After reading the response, I was wondering if I can get a plant DNA and then feed a protective agent and one sample without protective agent and see if the agents helped survive the DNA or it damages it. I don't have a calibrated X-Ray machine but I will figure out what to do so I can X-Ray it.

And also I am just wondering if I can use Polymerase Chain Reaction to see the damages. Also I wanted to know how I could connect this to life?

X-rays can produce harmful chemicals called free radicals that damage DNA, RNA and proteins. Antioxidants like vitamins C and E and foods like blueberries that contain antioxidants can destroy free radicals and prevent the damage. If you look in the supplement/vitamin section of a grocery store or pharmacy you can find many antioxidant preparations. You could choose several different ones to test to see which one is best able to protect cells from X-rays.

I would use yeast as the test organism and cell survival as the outcome. X-rays cause single- and double-strand breaks in DNA and you might be able to see this by isolating the DNA and running it on an agarose gel. As to whether you could use PCR--what sequence would you try to amplify? If you tried to amplify a DNA sequence with a break in it you would not get any amplification, but since you can't really predict which part of the DNA is going to be damaged, I don't see how you can use PCR.

X-rays can also modify DNA bases and these can be detected by immunofluorescence, but unless you have access to a university research lab and fluorescence plate reader, you won't be able to use this method.

You said that you don't have access to an X-ray machine. How do you plan to do this project without that?

X-ray-induced DNA damage that is not repaired can cause mutations and sometimes these allow cells to start growing and dividing without stopping--cancer, in other words. That is the connection between radiation and life. Most mutations cause no problems to a cell. In fact, the ability of a living being to evolve and adapt to environmental changes depends on mutations in the DNA.

Let us know if you have more questions.

Sybee

Thank you for the reply again. I am certainly interested in doing my experiment based on the ideas you gave me. As previously I said I don't have an X -Ray machine but after talking to my teacher I found an X Ray machine but I have to calibrate it. And otherwise I am starting my research now on the new ideas. Thank you once again.

I'm glad my advice was helpful. Keep posting as you have more questions and we will guide you.

Be careful with X-rays! They are harmful to human cells and lead shielding is necessary to block them. X-rays can penetrate walls and floors and you don't want to be irradiating someone whose office happens to be next door to the X-ray machine!

X-ray machines use voltages in the 15,000 to 25,000 V range which are deadly so be careful of that. If you have any questions about the operation of the particular model of X-ray machine, don't try to figure it out on your own--call the company that makes it and confirm the proper operating procedure.

Good luck!

Sybee

Thank you. And sure I will not try to do anything that will be dangerous.

Hello, I was just wondering how would I feed berries to yeast?

Just put the berries into a food processor or blender with some water and whiz them up. Use a coffee filter to strain out the bits. If you happen to have a juicer that would be the easiest thing to use.

What I don't know is how much juice to add to a certain amount of yeast. You should use a range of amounts of juice to test its effect. If it does increase longevity then you should see a steady rise as you add more juice up to the point where you have saturated the yeast.

You'll have to do some experimenting to find the correct proportions of juice to yeast to see the maximum effect--if there is one. Remember that juice contains a lot of different components, some of which, like acidity, might actually inhibit the growth of the yeast.

Let us know what happens.

Sybee

Thank you. I am also wondering in order for me to see if the radiation has damaged the DNA, I have to do gel electrophoresis for it right. Thank you once again.

Hi,

I had suggested that you just determine survival as a measure of X-ray damage but you can try extracting DNA, running it on an agarose gel and measuring the relative sizes of the bands. X-ray-induced DNA strand breaks will make the DNA smaller and run faster on the gel than intact DNA.

Have you ever done agarose gel electrophoresis before? Do you have the equipment and chemicals to run a DNA gel and detect the bands? I would recommend using Sybr-Green dye to detect the DNA because ethidium bromide is a strong mutagen and unsafe to work with as well as difficult to dispose of safely.

Did you succeed in getting the X-ray machine working? Keep us posted on your progress and we can help you troubleshoot any problems along the way.

Good luck!

Sybee

Thank you. So if I wanted to measure the survival of the DNA after the x-ray, I am wondering how will I measure it? And yes I have previously done agarose gel electrophoresis, and also my Biotech teacher as the equipment for it. Also my teacher said that he has a x-ray machine which is 95% done and I have to just order a part that's need. Thank you once again.

Electrophoresis is probably the easiest way to check the DNA for strand breaks caused by X-rays. Do you have a set-up for doing agarose gels? I would try a 1% agarose gel first using Tris-borate-EDTA buffer. Do some reading on Google scholar or PubMed and see if you can find an example of DNA run on agarose after ionizing radiation. It would be helpful to have an idea what you are looking for before you do the experiment.

Did you get the X-ray machine working? Do you have a way to accurately measure X-ray dose in grays?

Sybee

Thank you. Yes my teacher he does have a set-up agarose gels that I can use for my experiment. And I am still trying to get the x-ray machine working.

I cannot emphasize enough to be careful using an X-ray source. Always have someone else there to help you and make sure everything is done correctly and you and everyone is away from the area before the source is turned on.

I asked you this before--do you have a way to measure the X-ray dose? This is essential. If you use too low a dose you won't see any breaks and if it is too high you will totally destroy the cells.

Keep us posted on your progress.

Sybee

I am sorry I just don't understand what you mean by how will I measure the dose? Since I am just seeing which berries help protect DNA from x-ray radiation. Thank you.

Sorry for the confusion. The word 'dose' can have many meanings. What I am referring to is a measurement of the amount of ionizing radiation--X-rays--absorbed by a biological material such as DNA. You need to do some reading on radiation and X-rays. The wiki is pretty accurate although very technical: https://en.wikipedia.org/wiki/X-ray

When you got to the wiki, look at section 10, "Units of Measure and Exposure". This will explain what I mean by X-ray 'dose' better than I can.

Isn't there anyone at your school who is familiar with the X-ray machine you are planning to use? There are several different instruments that can be used to measure the X-radiation. You need to know this because the intensity of the radiation and thus how much damage it can do to DNA varies with the voltage on the X-ray tube. This is something that you control, so you need to be able to measure the X-ray dose that you get for a certain voltage. Then you need to adjust the machine so you don't give too high a dose.

I hope this makes sense now. Please, if you are not sure you understand what I am saying--ask. That is what this forum is for. We don't expect you to know all this technical stuff, but if you are going to X-irradiate cells then you will have to learn about it so you can explain what you did to the judges and why.

Keep posting and we will keep guiding you along!

Sybee

Thank you. Yes I understand now about the doses. I was just wondering how exactly does protective agents will help protect the yeast from radiation. While I was talking to my teachers regarding the question they said that protective agents could probably make a barrier on top of the DNA to protect it from radiation. So I was like does it really creates a barrier or there is something else. Thank you.

Good question!

Antioxidants are a sort of a barrier to radiation--but not a physical one. X-rays are powerful enough to ionize atoms which means they can add enough energy to the atom to rip off electrons. You remember that an atom in its elemental state is electrically neutral, right? The number of negatively charged electrons is exactly balanced by the number of positively charged protons.

X-rays can knock off an electron from the outer electron shell of an atom in a molecule and if you take away one negative charge then the atom has a net charge of +1--it's an ion or free radical. These ions can be highly reactive because they want to get their electron back and they will rip one away from any other molecule they get close to. They are dangerous things to have inside your cells. They can actually lead to single-strand breaks in DNA and RNA that can cause mutations and cancer.

Antioxidants are molecules that are specifically designed to interact with these ions and free radicals. They readily give up an electron and thus neutralize the radical before it can do damage--if they are present. By adding antioxidants to your cell cultures, some of the X-ray damage to DNA will be prevented and you should be able to see this on an agarose gel. The DNA without antioxidants should be smaller because the damage causes breaks in the DNA backbone.

Here's some of the references I used if you want to read more about ionizing radiation, radicals and antioxidants:

https://en.wikipedia.org/wiki/Ionizing_radiation
https://en.wikipedia.org/wiki/Radical_%28chemistry%29
https://en.wikipedia.org/wiki/Antioxidant

Keep asking questions and we'll help you to do a really good project.

Sybee

Thank you so much. I am just wondering can I use the comet assay technique to identify the DNA damage.

Hi,

The comet assay works very well to measure DNA strand breaks. Do you have the necessary equipment to do this kind of assay? It is technically more difficult than a simple agarose gel and works best when you have a fluorescence imager. I have never done the assay but I understand the method. If your teacher feels that you can use this technique then by all means do it.

Sybee

Hello,
My teacher said that I can use the technique. Thank You.

Excellent! Please let us know how well this assay works for you so we can use the information to advise others.

If you have technical questions as you go along, be sure to post them to us asap. In science, details are critical to the success of an experiment. Don't assume that you know how to do something without checking first. If something doesn't seem right, ask us about it. We may be able to save you a lot of trouble and wasted time.

Good luck!

Sybee

Hello,
I have slightly changed my science fair experiments. Instead of using X-ray radiation I am using UV radiation and instead using yeast I want to use peas DNA. So far I have planned to extract the DNA out of peas and then add the the berries to see if the berries have the power to protect the peas DNA from UV radiation. I have a question about how do I give the berries antioxidants to DNA. Thank you.

Hi,
I think UVB is probably a simpler choice than X-ray. Are you going to use the UVB reptile lamp?

I suggest that you also buy the UV test strips that I suggested before so you can measure the amount of UV that you are giving.

I'm not sure how to make the berry extract but you can do some research on it and find a formula. We can help you once you have a possible recipe. You could also buy some antioxidants and try those.

I'm sure that you will have questions so keep asking.

Sybee