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mwcrepeau@hawaii.rr.com 11-29-2005 08:50 PM

ladder pattern in gDNA extractions
 
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!


Michael Sullivan 11-29-2005 09:57 PM

ladder pattern in gDNA extractions
 
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, [Only registered and activated users can see links. Click Here To Register...] wrote:


---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
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Madison, WI 53706
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Peter Ellis 11-29-2005 10:20 PM

ladder pattern in gDNA extractions
 
[Only registered and activated users can see links. Click Here To Register...] wrote:

Isn't it simply apoptotic DNA laddering? The laborious pretreatment
could easily be leading to cell death.

Peter

Brian Ballard 11-29-2005 11:15 PM

ladder pattern in gDNA extractions
 
In article <[Only registered and activated users can see links. Click Here To Register...]> ,
Peter Ellis <[Only registered and activated users can see links. Click Here To Register...].uk> wrote:


That's exactly what it is; and quite a nice demonstration of it!

Brian


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