Science Buddies: "Ask an Expert"

Hi,
At first, I was doing labwork to find the SNPs caused by solar injury, in DNA extracted from someone's hair.
However, this didn't work very well, so I decided to do an ELISA.
The thing is, I started out doing a bioinformatics project and then wanted to do labwork. However, in the ELISA, I am testing for pyrimidine dimers, which are different from SNPs.
Is it possible to find pyrimidine dimers using bioinformatics methods?\

Thanks, blueswim

Hi,

I am not familiar with this topic, but here are a couple of abstracts that suggest that bioinformatics is useful in analyzing pyrimidine dimers:

https://www.cell.com/biophysj/abstract/ ... 08)70565-4

http://lib.bioinfo.pl/meid:2082

http://www.nature.com/emboj/journal/v24 ... 0849a.html

I am very impressed that you are doing lab work on this topic. This is a new area of research, so you might want to contact the primary authors of these papers with any additional questions you have.

Please let me know the details of your experiment if you need more help. I need to understand exactly what you are doing.

Donna Hardy

Hi,

I am very interested in the method you are using to measure pyrimidine dimmers for the ELISA assay. Do you have a protocol you can cite or attach to a reply?

Donna Hardy

Here's the procedure I'm using to detect the pyrimidine dimers:
1. Genomic DNA is denatured by heat and rapid chilling and added to polyvinylchloride flat-bottom microtiter plates precoated with 1% protamine sulfate (15ng of DNA for CPD detection, 150 ng of DNA for 6-4PP detection).
2. After drying in a 42° C incubator, the plates are washed with PBS containing Tween 20 and incubated with blocking solution to avoid non-specific antibody binding.
3. The plates are sequentially incubated with TDM-2 or 64M-2 antibodies specific for CDP or 6-4PP, respectively, biotin-labeled secondary antibody and finally streptavidin conjugated with horseradish peroxidase (HRP).
4. After washings, substrate solution containing o-phenylene diamine and H2O2 in citrate-phosphate buffer is added to the plates and incubated for 30 min at 37° C.
5. Absorbance at 490 nm is measured using a microplate reader after the addition of 2 M H2SO4 stop solution.
6. The amount of photolesions detected for each group will determine the correlation between sun exposure and damage.

It is described in
Akemi Nakagawa, Nobuhiko Kobayashi, Tsutomu Muramatsu, Yukio Yamashina, Toshihiko Shirai, Toshio Mori, Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei, Journal of Dermatological Science, Volume 16, Supplement 1, March 1998, Page S227,

Matsunaga, Tsukasa. "In Vitro Assays for Evaluating the Cellular Responses to DNA Damage Induced by Solar UV." Proc. 6th World Congress on Alternatives & Animal Use in the Life Sciences (2007). Print.

blueswim

Also, I think I may have figured out a little something to do for the bioinformatics portion. I'm not sure how to do it yet, but I have some idea of what I could try.
I'm thinking maybe, I can find the sequence of DNA with the pyrimidine dimers in it, and get other sequences with solar damage and then do a sequence alignment. I don't know if or where I can find these sequences, but if its possible then I can see what I can predict will happen for the lab work, which is what I wanted to try this year - since I've done a bioinformatics only project last year.

The question I'm investigating is "Do people with more sun exposure show more genetic damage associated with solar injury than those who don’t?"

blueswim

Ok, I have found the sequence for cyclobutane pyrimidine dimer which might be possible to be used in a sequence alignment with DNA:
http://www.ncbi.nlm.nih.gov/protein?ter ... ne%20dimer
And I THINK this one is the photoproduct 6-4:
http://www.ncbi.nlm.nih.gov/protein/NP_001081421.1
I searched "solar damage" at http://www.ncbi.nlm.nih.gov/protein which I think gives me protein sequences that have UV light damage.
I don't know where to find "normal" DNA though.

blueswim

Hi,

I apologize for the delay in responding, but this is not my area of expertise and it is going to take a little more time to send you a reply. I think you have made good progress on the bioinformatics part of the project, however. Are you still going to do the lab work? I'll get back to you as soon as possible.

Donna Hardy

Yes, I am still doing the lab work.
Okay, when I was looking for sequences I kind of came upon a problem.
I don't really understand what is the difference between a photolyase and one of the antibodies for the photoproducts.
I've tried doing research into the difference, but all I found were articles which made it more confusing.
If you can, please help.

Thanks,
blueswim

Hi Blueswim,

A photolyase is a light dependent enzyme that reversibly binds and repairs DNA damage and restores the pyrimidine dimmer back to the original base.

http://stuchebrukhov.ucdavis.edu/DNA_Repair/index.html

An antibody is an IgG molecule that specifically binds to the pyrimidine dimer and can be used to detect the pyrimidine dimers in an ELISA assay, since the binding coefficient is very strong.

http://www.ncbi.nlm.nih.gov/pubmed/1780359

Your ELISA procedure specifies using one of the specific antibodies. The binding of antibody is then detected by using a species-specific antibody to the primary antibody in a classic sandwich ELISA protocol and the color is developed using horseradish peroxidase with a colored substrate. Here’s a basic protocol.

http://www.ehow.com/how_5887403_perform ... assay.html

Are you having trouble getting a standard curve on your ELISA assay?

I spent some time searching for a Bioinformatics solution to your earlier question and I really could not come up with anything useful. I don’t think it is possible to search for pyrimidine dimers using Bioinformatics. For a science project, I think it might be better to start with work from a previous researcher and continue from there.

What is the overall purpose of your project and have you developed a hypothesis yet?

Donna Hardy