Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

DNA/RNA into nucleotides - (Feb/06/2009 )

I would like to digest DNA or RNA into individual nucleotides, is that somehow possible?

-Neumark-

Neumark on Feb 6 2009, 04:54 AM said:

I would like to digest DNA or RNA into individual nucleotides, is that somehow possible?

Why don't you just buy these? But I guess you may have some reason. I can conjecture that treating with DNAse and RNAse would do the trick, but not sure how complete the fragmentation would be to give you mononucleotides.

Let us know when you figure it out.

-cellcounter-

cellcounter on Feb 6 2009, 06:04 PM said:

Neumark on Feb 6 2009, 04:54 AM said:

I would like to digest DNA or RNA into individual nucleotides, is that somehow possible?

Why don't you just buy these? But I guess you may have some reason. I can conjecture that treating with DNAse and RNAse would do the trick, but not sure how complete the fragmentation would be to give you mononucleotides.

Let us know when you figure it out.



I would like to detect the amount of an incorporated drug into different viruses. Sequencing will not do the trick, since it will be recognized as a normal nucleotide.

It feels like a chemical would do the trick, but my chemistry knowledge is not that great.

I wonder if DNAse and RNAse would work, it does not feel like they do a complete fragmentation. I will have to read up on that.

-Neumark-

It's still unclear to me what you're trying to do -- say you can digest to individual nucleotides, what would you do with them?

-HomeBrew-

do you just want to know the quantity of the compound integrated into the polynucleotide sequence? Or do you need to know its location too?

-perneseblue-

DNase and RNase digestion will result in generation of oligomers of different sizes, not what you seem to want.
In the past I have been determining drug incorporation into DNA from patients on thiopurine therapy. Digestion of DNA with P1 nuclease (Roche) will result in full release of deoxyribonucleoside 5-monophosphates.

To 100 ul (1-5 /ug DNA) add 10 ul of digestion buffer (500 mM sodium acetate buffer-10 mM MgCl2, pH 4.5). Then add 20 ul of enzyme
stock solution (25 ug/ml P1 nuclease, 12.5 units/ml acid phosphatase in 50 mM sodium acetate buffer-1 mM MgCl2 , pH 4.5). Incubate 1 hour at 42 oC.

The resulting deoxyribonucleosides can be analysed by reversed phase HPLC on C18 columns. If you want deoxyribonucleoside 5-monophosphates (for ion-exchange analysis) omit the phosphatase treatment.

The method is fine for RNA too. I usually heat denature the DNA before digestion, but this may not be necessary if certain DNA extraction methods are used.

Hope this is helpful.

-klinmed-

Yes, I only want to detect the amount of the incorporated drug. Nothing more and nothing less.

I will try the advice by Klinmed (thank you very much) and I will let you guys know if it worked or not.

You might read about it in the May edition of Science :blink:

-Neumark-