1
$\begingroup$

if one wanted to compare how sources of DNA would degradate in various conditions(environmental) how would they do this in an experimental fashion but also be best comparable to real world examples of Enviromental conditions? Sorry if this question is worded poorly, any suggestions would be appricated.

For example, if I were to expose my own blood to various conditions such as a wet enviroment versus a dry ennviroment, how would I best replicate these enviroments in real life(ie: bloodly water in a cup versus dried blood on a floor) without introducing confounding varables?

I would need some type of "standard" yes? And if so, then would using sterile controled conditions be appropriate, or should I literally be using these real world situations for "better" results?

I tired finding papers, but I guess my research methods are too poor to get anything useful.

EDIT:

My facilities I have access to are a small academic institution, I know basic autoclave use and have access to gel rigs and pcr machines. My samples do not have to be blood, it could be any human tissue or sample, like hair or spit. I suppose I would use the same sample, but kept in a sterile condition for a controo, maybe a petri dish?

I guess my mental protocol would be to try different degradation conditions and extractions, but starting with a boiling DNA extraction to see how effective a certain condition was compared to a control.

I could set up a time table of like 2 weeks to perform the experiment, and every 3 days or so, extract DNA from the control and the sample to then pcr with some human primers(I think we are using the fbi's CODIS primers that they use in foriensics, but only a subset due to cost) and then run an agarose gel to determine degradation( poorer resolution meaning more degradation I suppose?).

Another wuestion that popped to mind, for my time table and my DNA extractions, would I have to perform the pcr and subsequent gel elecrrophorsis as soon as I am done with my extraction, or could I store the extraction samples until I am ready to run a large batch of pcr tubes and a large gel? Would they degrade in a -80°C freezer?

$\endgroup$
  • $\begingroup$ You are going to need to add more details to your question. What kind of facilities to you have access to? If you are doing this in an academic or commercial lab, there are likely protocols for handling human tissue that needs to be followed. Human blood is a biohazard and there are likely strict rules in place as to what you can and cannot do. Also, what is your plan for processing and examining the DNA after your experimental time. PCR amplification, sequencing? What are your controls? Until you give these details, then it is hard to say. $\endgroup$ – AMR Dec 26 '15 at 3:59
  • $\begingroup$ @AMR. I added more details, thanks for the suggestions. This is an academic lab, I have access to autoclave, pcr, gel rigs etc..(we are not rich, state school). I was using blood as an example, there are other tissues I can use, although I can get authorization for blood if needed. Controls would be a sample of tissue or product that is kept in sterile conditions, like a perri dish maybe? Or would that be a bad idea. A lot more detail is in my post above. $\endgroup$ – Ro Siv Dec 26 '15 at 12:49
  • $\begingroup$ Since you mention examples, a colleague did a project on DNA degradation using cotton swabs with saliva (definitely not as hazardous as blood!). What I don't know for sure is if she used normal PCR, but I do know she didn't have access to qPCR (and she was really good at agarose gels!). My bands were never perfectly regular, though, and I was just testing for presence or absence. $\endgroup$ – Yisela Jan 15 '16 at 16:33
2
$\begingroup$

Have you heard of qPCR? This method quantifies the amount of DNA in the sample. Another quantitative PCR method you might look into is taqman PCR. Traditional PCR + agarose gel electrophoresis is not going to give you a quantitative measurement of the DNA in your sample. Here are the wikis on taqman and qPCR.

taqman https://en.wikipedia.org/wiki/TaqMan

qPCR https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction

Also, if you have the choice between intercalating agents and fluorescent probes, go with fluorescence. Intercalating agents are hazardous.

Hope that helps.

Edit Adapted from comments -

We don't have access to a real time pcr intrusment sadly. All we have is a traditional pcr machine. Can I get your opinion on this quote I found on the quantitative messures of traditional pcr: '"No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you 'semi-quantitative' results." From thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/…. Are there ways to work with the intensity of the bands I get to be " more reliable"? – Ro Siv

Yes, quantification from traditional PCR can be done, though the accuracy is questionable. One method I have heard of uses a computer to analyze the intensity of the bands. You could probably pull this off with some Python code (check out the scikit-image python library). The other option is to do what you said, compare to known concentrations.

If you are familiar with HA titers in virology, you might be able to contrive a similar method, in which you carry out 10-fold dilutions of the sample until there is no more detectable DNA left. The point at which there is no DNA left is called the experimental end-point, and the titer of the original sample is simply the inverse of the dilution factor at the end-point. Check out hemagglutination(HA) in the context of virology. I think a method similar to this would be better than simply eyeballing the intensity of bands, because you will have some quantitative evidence.

If you want to eye-ball, you should use control bands at several different concentrations, so that you can say, 'the intensity of the band falls between control bands A and B, and so the concentratin of the sample is between concentrations A and B'

I bet you could pull off the serial dilutions. It will be good practice, too. To make a 10 fold dilution, just add 1ml of sample to 9ml of saline. Then, from that new solution you just made, do it all again. Take 1ml and add to 9ml saline. Repeat. Each time you decrease the concentration by 10^-1. You can scale it down too - 1ul sample to 9ul of saline. Repeat. Here is a SOP on HA. Honestly, I would go with the serial dilution thing. I bet ur professor would help you out with this. It is cheap, easy, and widely used. cfsph.iastate.edu/HPAI/resources/SOPs/HA_HI.pdf

Question: If I were to do both methods, then would ImageJ be a possible substitute for scikit-image? rsb.info.nih.gov/ij I think its GUI interface is alot more workable for me.

Response: From just skimming that link it looks like it might. You would probably be interested in the density analysis part of the software, but I dont think a computer is necessary, and ya you will have to produce that gradient of concentrations any way. If it were me, I'd first try to do the experiment without computer analysis. Produce the gradient of control DNA concentrations, compare your sample to the control gradient and see where your sample falls.

Then repeat the experiment while varying the independent variable, lets say its time. So take a sample at 5 min 10 min 15 min etc. and produce a plot of surviving DNA concentration vs time. It will probably end up looking something like a sigmoid curve. If you decide you need more precision in your measurement of the DNA concentration, then consider using the image software, but I doubt you will need it, though I admit it would be twice as badass.

$\endgroup$
  • $\begingroup$ We don't have access to a real time pcr intrusment sadly. All we have is a traditional pcr machine. Can I get your opinion on this quote I found on the quantitative messures of traditional pcr: '"No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you 'semi-quantitative' results." From thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/…. Are there ways to work with the intensity of the bands I get to be " more reliable"? $\endgroup$ – Ro Siv Dec 26 '15 at 20:46
  • 1
    $\begingroup$ Yes, quantification from traditional PCR can be done, though the accuracy is questionable. One method I have heard of uses a computer to analyze the intensity of the bands. You could probably pull this off with some Python code (check out the scikit-image python library). The other option is to do what you said, compare to known concentrations. $\endgroup$ – DonJulian Dec 26 '15 at 21:16
  • $\begingroup$ If you are familiar with HA titers in virology, you might be able to contrive a similar method, in which you carry out 10-fold dilutions of the sample until there is no more detectable DNA left. The point at which there is no DNA left is called the experimental end-point, and the titer of the original sample is simply the inverse of the dilution factor at the end-point. Check out hemagglutination(HA) in the context of virology. I think a method similar to this would be better than simply eyeballing the intensity of bands, because you will have some quantitative evidence. $\endgroup$ – DonJulian Dec 26 '15 at 21:16
  • $\begingroup$ If you want to eye-ball, you should use control bands at several different concentrations, so that you can say, 'the intensity of the band falls between control bands A and B, and so the concentratin of the sample is between concentrations A and B'. $\endgroup$ – DonJulian Dec 26 '15 at 21:16
  • 1
    $\begingroup$ The extended conversation that pertains to the answer should be inserted into the answer as an edit. Comments are temporary and can be lost in time. To insure the information stays with the answer it should be added to the body of the answer. Please consider editing to add the information into the answer. Thanks, $\endgroup$ – AMR Dec 26 '15 at 23:47

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.