Mitochondrial isolation for mt DNA extraction from Earthworms

Dear collegues
I wonder if anyone ever tried to isolate mitochondrias from earthworms
for posterior mtDNA extraction, could anyone send me a good protocol?
I am having a lot of trouble doing that, probably due to soil
contamination in the digestive tract, even after depuration and I am
getting very low yield of mtDNA. Any help?? I am working with
Pontoscolex corethrurus, my model organism .

Best regards

Am 05.10.2009, 02:48 Uhr, schrieb luiscunhamx <[Only registered users see links. ]>:


For histology, it is customary to feed earthworms for two weeks on humid
filter paper pieces, which are changed daily. That removes any soil from
their digestive tract, which would otherwise nick the microtome knife.

I am not really sure whether this reply will help or not, I used to work
on C. Elegans. As part of that work one of the topics we touched upon
was worm metabolism. When certain deep soil worms (worms that live in
deeper anoxic layers of the soil or in wetlands) are exposed to low
oxygen environments they tend to shut down classic oxidative catabolism
and revert to a low oxygen using pathway (mammals don't have this
pathway). Mitochondria respond to signals and may decrease in number
when challenged. One possible way to increase mitochondrial DNA is to
increase mitochondria by feeding these worms a preferred substrate in a
better oxygenated environment.

Hope this helps, though doubt it will.

BTW, despite the fact that mtDNA has a higher rate of mutations there
are some highly conserved regions of mtDNA, I did a study of all the
known therian mtDNA sequences and found something >4000 sites that have
not evolved in any of the sequenced mtDNA. You should be able to amplify
using PCR between these highly conserved regions with a high fidelity
polymerase like Fusion, but you will need a clean source of DNA. Since
mtDNA is circular you might want to first nick the mtDNA trying
different rare cutting endonucleases that cut these conserved sites from
known mtDNA sequence within the local clade. At 17,000 nucleotide you
should be able to amplify within 8.5 minutes the entire mitogenome. IOW,
you should be able to isolate DNA from single cells, or washed egg
cells. Oh and one other thing, since mtDNA is circular (I assume it is
circular in this species) it will be more difficult to isolate than
autosomal DNA strands, it will none the less precipitate, if you are
using a DNA isolation kit, cool the isopropanol mixture down to 4'C and
let the DNA settle down for a few hours before centrifugation, and be
careful the DNA may not be in a concise pellet, but spread around the
bottom of the tube. Sonication, if used in the preparation will produce
random breaks in the DNA and make it linear.


-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of luiscunhamx
Sent: Monday, October 05, 2009 1:48 AM
To: [Only registered users see links. ]
Subject: Mitochondrial isolation for mt DNA extraction from Earthworms

Dear collegues
I wonder if anyone ever tried to isolate mitochondrias from earthworms
for posterior mtDNA extraction, could anyone send me a good protocol?
I am having a lot of trouble doing that, probably due to soil
contamination in the digestive tract, even after depuration and I am
getting very low yield of mtDNA. Any help?? I am working with
Pontoscolex corethrurus, my model organism .

Best regards
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