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!