DNA Adducts

[Frexx]

Need serious help: Can DNA adducts be measured quantitatively by HPLC-UV alone? (Aflatoxin-DNA adducts in particular)

 

What are other methods that I could use in measuring Aflatoxin-DNA adducts that are not so expensive yet will yield reliable results?

 

Thanks:-)

[scicop]

HPLC UV is just a purification system. You would need a combination of NMR and (perhaps) a MS machine (for mass calculations) for any form of structure determination.

[BioWizard]

There is a method described in this paper which might be helpful:

 

http://www.pnas.org/cgi/content/full/98/25/14601

 

Scicop, what you said is true, but HPLC can indeed be used for quantitative analysis as well.

[zyncod]

I've used the comet assay in the past as a genotoxicity measure. It's easy (if fairly labor intensive in the measuring stage), and you can probably use the photolability of aflatoxin adducts as a way of amplifying the specificity for aflatoxin in particular.

[Frexx]

Hi! Thank you so much for replying . I'm actually working on quantitative analysis of aflatoxin-dna adducts in the liver of sprague-dawley rats to test the chemopreventive property of a particular drug that is believed to inhibit its formation process.

Literature tells me that HPLC with fluorescence detector, accelerated mass spectrometry, radio isotyping etc. are common means to examine afla-dna adducts but these are not available at hand and very expensive too. I was trying to come up with a much simpler method yet will yield reliable results that will convince my panelists of the credibility of my data.

 

I'm a chemistry major by the way but my work is in biochemistry-related. My adviser wants to see chemistry in my work. I will have to defend my work to three panelists after to get my masters degree.

 

Scicop: yup... HPLC-UV is primarily used only in so many other experiments as purification system for the adduct. However, due to limited resource, i was thinking of using it to quantify my adduct as well. I think this is possible if I have set of standards to compare with anyway. But can this method stand?

 

Biowizard: Hi.. I checked the paper already. The methods were alright except that they used urine samples for the analysis. Problem is I am dealing with liver samples

 

Zyncod: Yup comet assay! Actually, COMET assay was my initial plan since it can detect presence of DNA damage. Indeed, its method is quite simple... but my adviser made me focus on quantifying the adduct first... *sigh*

 

Guys, thanks soooooo much!!!!! I'll wait again for your replies!

[scicop]

Well, if you have standards and you can show that your adduct containing DNA elutes at a corresponding peak of your standards then it can be a possiblity, although you will have to determine a detection range using various concentrations of your standards. The key would be having sufficient controls. Make sure you have controls to support the validity of your approach.

 

One quick way to do it is if you have standards or a purifed form of your adduct (chemically synthesized) you can make an antibody. Keep it simple and immunize a rat. You can get yourself a rather dirty poly-clonal ab within a couple months that will allow you to detect your adduct, although you'll need the appropriate controls as well. But, again, your ability to detect anything will depend on the concentration of your DNA adduct in the liver, that is, how much damage is your mutagen really causing.

[mattbimbo]

you could try an antibody based method for detection of DNA-aflatoxin adducts.