dsDNA and ssDNA

Barbara MacGregor via methods%40net.bio.net (by bmacgreg from unc.edu)
Fri Jan 2 13:58:32 EST 2009


>
>
> From: "Paul J. Phelan" <Paul.Phelan from tufts.edu>
> Date: January 1, 2009 6:31:05 PM EST
> To: methods from magpie.bio.indiana.edu
> Subject: Electrophoresis of dsDNA and ssDNA
>
>
> I have a simple question that so far does not seem to have a simple  
> answer.
> I need to confirm that a 60-mer dsDNA oligo is double-stranded.
> This is poly-dA/dT DNA.  Does anyone have a straightforward 1D  
> electrophoresis protocol that will separate single-stranded and  
> double-stranded forms of an oligonucleotide in the same range as a  
> 60-mer oligo?
> I have already been surprised to learn that in 7 M urea PAGE, ssDNA  
> will run SLOWER than dsDNA.  Does anyone have experience with this?
>
> Thanks in advance,
>
> Paul
>
Hi,

You might try digestion with enzymes specific for ss or ds DNA - for  
example, E. coli Exo I is specific for ssDNA. Somebody should be able  
to spare a little... That's probably the simplest way.

If you have the ss oligos, you could run them side by side with the  
putative ds oligo on e.g. the 7M urea PAGE gel, hopefully things would  
line up nicely.

You could also try running the sample with and without heat  
denaturation, to see if extra bands appear, but that might not give an  
unambiguous result. As a control I suppose you could try generating  
restriction fragments of the same approximate size from some  
convenient DNA, run them with and without heat treatment - although it  
would probably take somewhat higher temperature/longer incubation to  
denature DNA in general than your all-AT oligo.

Okay, end of random ideas, maybe someone has an easier answer.

Barbara MacGregor


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