We are trying to analyze formation of DNA-adducts through HPLC. Can anyone help us regarding the protocol to do DNA HPLC. In addition, can we run the complete human genomic DNA in HPLC column? If no, then what changes would be needed to detect DNA adducts through HPLC; If yes, then which column would be suitable and how the DNA should be processed to avoid clogging in the column. Suggestion regarding the composition of mobile phase related to DNA HPLC would also be highly appreciable.
Hi Atanu,
usually, DNA is hydrolyzed/digested into its nucleosides, which are then analyzed with HPLC methods. (few links attached). I think this is the only way to go for you. DNA is too complex/big for common reverse phase HPLC (and if you could do so, you may not be able to see any difference in the elugrams, since few modifications on the DNA strand will not alter the hydrophobicity much).
Recently, there was a report on size exclusion chromatography on DNA nanocages, but there you have distinct species which could be separated well (3rd link).
Good luck,
Marcus
https://academic.oup.com/femsle/article/25/1/125/570395/Determination-of-DNA-base-composition-by-reversed
http://www.biochemj.org/content/318/1/21
http://www.sciencedirect.com/science/article/pii/S0168365916305156
https://link.springer.com/article/10.1023/A:1014268729658
Article Measuring oxidative damage to DNA; HPLC and the comet assay compared
Thanks Marcus for your suggestions and the links. They were really very helpful.
Just another thing I wanted to know that the column that we have is C18 column with pore size of 120 Angstrom. If we use a column with pore size 200 angstrom can we still run genomic DNA through it?
I guess yes, the pressure may be a bit lower as well as the resolution, it's worth to try. We usually use Phenomenes Luna 100 Angstrom 5 µm particle colums (see link).
Our standard column has the article number 00G-4252-Y0 (2nd link). Phenomenex offers many nice features, such as the security guard cartridge kits (pre-columns with replaceable filters).
Marcus
https://www.phenomenex.com/Products/HPLCDetail/luna
http://www.phenomenex.com/Products/Part/00G-4252-Y0
If DNA adduct is our prime focus then I guess we should not compromise with resolution. I still feel that whatever we do, we should not run the complete genomic DNA through HPLC column.
Firstly, the adduct will be a very small molecule (an oxygen molecule or a methyl group) that will get covalently attached with the DNA. A high precision will be required to resolve this peak from rest of the DNA.
Secondly, the genomic DNA itself may have so many variations within itself which, if run through column, will result into background noise so huge that one cannot resolve the peak obtained due to formation of adduct.
Please rectify me if I have mistaken somewhere.
Hi Atanu,
you are right, especially if you modify only with one Methyl group... I guess it is impossible to separate this from the unmodified educt. You have to consider the mechanistics of HPLC - molecules get separated based on the strength of interaction with the stationary phase. If the difference between the forces is very little, there will be no separation occuring. I don't know if there are any other methods to determine the degree of modification for your case related to HPLC.
Your second consideration can be neglected if you denature the DNA before analysis and make every molecule "equal" (Urea, Guanidinium chloride? I don't know if this is possible).
The methyl group may change the conformation of the DNA - try x-ray diffractometry measurement? But still, the product may be not pure enough, a mixture of the modified and unmodified DNA...
Sorry,
Marcus
Atanu@ yes you can increase the pore size for higher molecular weight compounds. But you have to consider this aspect too while increasing Pore size the surface area will decrease. When decrease surface area resolution also will decreases.
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