ladder pattern in gDNA extractions

Michael Sullivan mlsulliv at wisc.edu
Tue Nov 29 16:57:24 EST 2005


Could it be apoptosis? One of the hallmarks of apotosis is formation  
of this sort of DNA ladder. It sounds like the cells go through a lot  
prior to the DNA extraction. Is it possible that all the handling has  
induced programed cell death? Are the coral cells viable if you  
purify them but don't extract them for DNA?

Just a thought.

Mike


On Nov 29, 2005, at 2:50 PM, mwcrepeau at hawaii.rr.com wrote:

> I've posted a gel photo at http://home.hawaii.rr.com/ovenfish/gel.jpg
> that I'd like some comments on.  Lanes 1 and 5 contain a 100bp DNA
> ladder.  Lanes 2 and 3 are linearized cloning vectors.  Lanes 6-8 are
> genomic DNA extracted from purified coral cells using the Qiagen  
> DNeasy
> kit.  Note the ladder pattern in these lanes.  Also note that the
> samples in lanes 2 and 3 were mixed with the same loading dye, so the
> loading dye is not contaminated with any DNA ladder.
>
> My interpretation is that we have inadvertantly repeated the classic
> experiment by Hewish and Burgoyne that demonstrated the regular
> periodicity of DNA packaging into nucleosomes.  The bands have a
> spacing of approximately 200bp which is consistent with selective DNA
> digestion by nucleases at the more vulnerable regions between
> nucleosomes, yielding concatamers of one nucleosome, two nucleosomes,
> three nucleosomes, etc. on up the ladder.
>
> We have seen this phenomonon occasionally with other species as well,
> including in extracts of EDTA-preserved whole blood from a marine
> mammal and frozen muscle tissue from a marine snail.  Some people in
> the lab have suggested that our instruments or reagents may be
> contaminated with nucleases, but extractions of other samples with the
> same instruments/reagents often yield high MW DNA, casting doubt on  
> the
> contamination theory.  Alternatively, endogenous nucleases within the
> cells of the tissue sample may be at fault.  The DNeasy protocol  
> begins
> with a lenghty incubation in the presence of proteinase K.  Perhaps  
> the
> digestion conditions lead to a liberation of the DNA from the nuclease
> and it is subsequently attacked by cytosolic nucleases before they
> themselves are inactivated by the proteinase?
>
> My questions are these:
>
> 1.  Have others seen this ladder pattern in their gDNA extractions?
> 2.  Does my "Hewish and Burgoyne redux" interpretation seem plausible?
> 3.  Could endogenous enzymatic activity be at fault?
> 4.  Can anyone offer suggestions on how to prevent the DNA  
> degradation?
>
> For the coral work the cells were laboriously collected and  
> purified by
> density-gradient centrifugation and flow cytometry in order to
> eliminate intracellular algal symbionts present in the species.  We  
> had
> hoped for high MW gDNA to use as starting material for microsatellite
> library construction, so the results were a little disappointing.
>
> Thanks in advance for your comments!
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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