Measuring Pyrimidine Dimers in Planaria

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mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Measuring Pyrimidine Dimers in Planaria

Post by mserrano »

Please refer to the below post to see my question involving the effect of UV rays on planaria. I look forward to your responses, thank you.
Last edited by mserrano on Tue Oct 20, 2015 9:05 am, edited 3 times in total.
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Measuring effect of UV Rays on planaria

Post by mserrano »

I am doing a project that involves observing the effect of UV rays on the planarian's ability to regenerate into two separate worms after being cut in half. I will be cutting a group of about 2 or 3 planaria in half, placing them in a petri dish with spring water, and, through the use of a biological safety cabinet with a UV light, will expose them to UV C rays. My question is, how can I measure the damage on the planaria? I will observe their ability to regrow after being exposed, but I am still searching for different ways to measure how the UV rays affect the planaria.
SUS
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Re: Measuring effect of UV Rays on planaria

Post by SUS »

Hello!

What a wonderful idea for a project! If I am not mistaken, it seems that you want to see if UV rays will affect the ability of the planaria to regrow. Therefore, you could measure aspects of regeneration to measure the affect of UV rays. For example, you could measure the growth rate (how much each planaria has regrown every day), or just measure total regrowth after a set number of days. You could also measure how long it takes to regrow into a complete worm after exposure.

One potential issue that may occur is that planaria can undergo lysis when exposed to UV rays. This would make it difficult to asses the affects of UV rays on regrowth, but would rather tell you the UV rays affect on planaria survival. To counter this, try several exposure times to find one that doesn't kill the planaria, but would allow you to measure regrowth. Hopefully this helps!

-Surya
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Re: Measuring effect of UV Rays on planaria

Post by mserrano »

Thank you so much! I didn't consider the fact that the UV rays might be too strong, and it is a fantastic idea to test at several different exposure times/strengths. I also considered exposing them to a very similar amount of UV rays that we experience when exposed to the sun to observe the effect of UV rays from the sun on our skin cells and their ability to regrow. Thanks again!
SciB
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Re: Measuring effect of UV Rays on planaria

Post by SciB »

I just read your reply about using a different wavelength of UV in your experiment and I think that is a good idea. The UVC that comes from a germicidal lamp such as the kind used in a biosafety cabinet to sterilize the interior is very intense and is at a wavelength that is known to cause a lot of DNA damage and strand breaks. Nearly all of the UVC in sunlight is absorbed by the earth's ozone layer so people or planaria are not exposed to it. UVB, however, does penetrate the atmosphere to a certain extent and has been associated with human cancer.

I would suggest that you get a lamp that emits UVB and use that to irradiate the planaria. These are not expensive and you can get them at Petsmart (http://www.petsmart.com/reptile/heating ... tid-500011). You could expose one group of planaria to UVB for different lengths of time while they are regenerating and the control group to a compact fluorescent lamp.

As Surya said, your main measurement should be how fast the planaria regrow their damaged bodies. Your hypothesis could be either that UVB makes them regenerate faster or slower. There will biological effects from the UVB on the planaria but these would not be so easy to measure. You would need some advanced instruments and test kits to do such measurements and those are expensive.

I hope this suggestion helps. Please post again with more questions.

Good luck,

Sybee
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Re: Measuring effect of UV Rays on planaria

Post by mserrano »

Thank you so much for your reply! I will buy the UVB Lamp, and will proceed to follow your suggestion with the test group and control group. One question though, what is the reason for using a fluorescent lamp for the control group? And what is the benefit of using the fluorescent lamp rather than just allowing the planaria to grow without any external disturbances?


Thanks again,


Madison
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Re: Measuring effect of UV Rays on planaria

Post by SciB »

Hi Madison,

When you run an experiment there is always a 'control' that is treated exactly the same as the others except that the test treatment--the UVB in your case--is omitted. Ideally you would use a lamp exactly the same as the reptile lamp except that it does not produce UVB. I don't know where you would get such a lamp, so I suggested using a plain compact fluorescent bulb that does not produce UVB.

You should expose all the planaria to the same number of hours of light and then cover them so that they are all in the dark for the same length of time. The light is your independent variable and you need to control it carefully.

If you have more questions, please let us know. Planning a project carefully is as important as doing it correctly.

Sybee
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

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Post by mserrano »

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Last edited by mserrano on Tue Oct 20, 2015 4:39 pm, edited 4 times in total.
dcnick96
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Re: X- Ray radiation effect on the regenerative cells of Planari

Post by dcnick96 »

Hello. Please keep all questions for your project in the same post, so that our experts can respond appropriately based on what has already been discussed. Good luck, and thanks for using Science Buddies!
Deana
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Measuring DNA damage in planaria

Post by mserrano »

What is the most effective way to measure DNA damage in planaria that have been exposed to UV?
SciB
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Re: Measuring DNA damage in planaria

Post by SciB »

Before we can answer your question we need to know if you have access to a research lab with a person to help and supervise your work. There are several ways to quantitate DNA damage but they require expensive instruments and reagents not usually available in most high school labs. We can suggest the best methods but if you don't have the resources or time to do them then it won't help you.

Did you do a search for UV damage to DNA? If so then you know that the most common damage from UV is the chemical bonding of two adjacent thymines to form a dimer. https://en.wikipedia.org/wiki/Direct_DNA_damage

This damage blocks transcription, so a gene with T-T dimers cannot be expressed. Cells do have enzymes to repair these dimers, however, and I expect planaria is able to repair UV damage up to a point.

Here is one of the best methods for detecting thymine dimers: http://aem.asm.org/content/73/3/947.full

From your question I assumed that you wanted to measure the damage itself in the planarian's DNA after UV. Is that what you meant or did you want to know how to measure the effect of DNA damage on reproduction, regeneration, memory or some other behavioral or physiological function?

Post again with some information about your resources and tell us what your hypothesis is and we will be able to answer your questions better.

Sybee
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Re: Measuring DNA damage in planaria

Post by mserrano »

Hello! Thank you so much for your reply!

Yes, I would like to measure the damage in the planaria's DNA after UV, but I would like to use this information to measure the effect of that DNA damage on the planaria's regeneration. After cutting them in half and exposing them to the UV, I would like to compare the test subject's regenerative abilities (how much they regenerate over a certain amount of days after prolonged UV exposure) to the regenerative abilities of planaria that were not expose to the UV (control). By comparing the DNA of the test subject to the DNA of the control, I would like to try and explain and formulate a reasoning for the physical observations and measurements in the planaria's regeneration after UV.

I am performing this experiment in my ROP Biological Sciences and Lab Research class, and our program is willing to provide us with the resources we need. So I will be able to send samples of DNA to a research lab for them to process the planaria's DNA and send the results back to me, but I was wondering if there was any simpler way I could maybe process or analyze the DNA in my high school lab. We have a variety of instruments in our classroom such as the resources and skills needed to perform gel electrophoresis, PCR, Column chromatography, etc. My teacher had an idea that I could maybe take a sample of the test subject and control planaria, and observe their DNA through gel electrophoresis. I am very intrigued by this idea and I would like to know your opinion on it and whether or not you believe this will be an effective method. I would also like to know of any other suggestions you may have.

Thank you so much, you have been a very big help and I look forward to your reply.

Madison
SciB
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Re: Measuring DNA damage in planaria

Post by SciB »

Hi Madison,

Wow! It sounds like you have the resources to do a really great project.

One thing I thought about while reading your proposal is the timing. You are giving the UV dose after dividing the planaria. What is your reasoning for doing it this way rather than giving the UV just before, or even a couple of days before, the regeneration part? The surgery will be a stress on the animals and maybe they will be more susceptible to the UV damage given afterwards. I don't know, but it is something a science fair judge might ask you about, so be prepared with an answer.

That's great that your teacher is suggesting a gel electrophoresis method to identify T-T dimers in DNA. You can learn so much more by doing the experiments yourself rather than sending the DNA to a lab for analysis. When I was in college we had to do most of the assays and tests ourselves because there were no kits to use much less a lab to send the samples to. I have much better knowledge about these tests because I did them myself and I can see ways to improve or change the commercial products or troubleshoot them when they don't work right.

OK. How can you detect UV damage in planaria DNA using gel electrophoresis? I did a search for how to do this and came up with one paper that I think is helpful: http://www.sciencedirect.com/science/ar ... 9508001045
The key to identifying the T-T dimers is to use an enzyme called T4 endonuclease V which cuts DNA at UV-induced thymine dimers. DNA that has been cut by T4 endoV is smaller than uncut DNA and the size can be measured by gel electrophoresis.

In the paper they used atomic force microscopy to measure the DNA but I think you could do it using either an agarose gel or a polyacrylamide gel. Ask your teacher if your lab has the capability of doing polyacrylamide gel electrophoresis. I don't know which type of electrophoresis you would need to use. It depends on the size of the DNA fragments. Agarose gels separate DNAs in the range of 200-100,000 base pairs while PAGE can separate DNAs as small as 10 base pairs.

Do some more reading about how to detect UV-induced pyrimidine dimers and see what you can come up with in terms of the resources that you have. PCR would work if you had a sequence that you knew had T-T dimers because the Taq polymerase would not be able to copy it; but you really need a method that allows you to measure dimers in the entire genome.

If I think of another way to do this experiment, I will let you know.

Good luck!

Sybee
mserrano
Posts: 13
Joined: Mon Sep 07, 2015 10:13 pm
Occupation: Student: 12th grade
Project Question: I am conducting an experiment involving xrays and their effect on the planarian flatworm, who has regenerative abilities. I'll be using an xray machine I will zap the worms and see how it affects their ability to regrow into two separate worms. I am doing this experiment with a love for the science fiction like nature of the worms and xrays. My biggest problem in developing this project is trying to find out how it will be innovative and helpful to people, and any feedback is helpful.
Project Due Date: March 2016
Project Status: I am conducting my research

Re: Measuring DNA damage in planaria

Post by mserrano »

Hi, thank you again for your reply!


I think your idea about the T4 endonuclease V is incredibly helpful, and I am very interested in pursuing it in my project.

I did have a question about the electrophoresis processes though. How will I extract the DNA from the planaria? I thought I could just take the planaria, crush them, add the T4 endo V, and run it through the gel. But my teacher said that just taking the planaria and crushing them might be too much information for the gel to process, overloading it. I have found a few other articles involving the processes of lysis to extract specific DNA, but I do not know what specifically I am looking for. Your insight on this problem would be extremely helpful, and I look forward your reply.


Thank you so much,

Madison
SUS
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Re: Measuring DNA damage in planaria

Post by SUS »

Hello!

I found an extraction protocol from MIT that details how to obtain DNA from planaria. Follow only section "a".

https://biology.mit.edu/sites/default/f ... ysis-2.pdf

One thing to definitely make sure is if all of these reagents are available to you. Secondly, I would compare this protocol with the others you might have found to see which may be easier to do. One way to test which protocol may be best is to run a small amount of the extracted DNA through a gel, and see which gives you a good yield.

Just a quick word of advice for your T4 endonuclease V reaction, use only a small amount of your extracted DNA rather than the whole thing. This way you may be able to see the bands clearer on whichever electrophoresis method you end up using. Hope this helps!

Regards,
Surya
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