X-ray and DNA

[SciB]

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

[skrish16]

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.

[SciB]

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

[skrish16]

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.

[SciB]

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

[skrish16]

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.

[SciB]

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

[skrish16]

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.

[SciB]

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

[skrish16]

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

[SciB]

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

[skrish16]

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

[SciB]

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

[skrish16]

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.

[SciB]

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