DNA degradation -- summary

[Kathryn Kavanagh]

Dear Listmembers-- 
Awhile back I asked for ideas about problems we had with degraded DNA from
the pomacentrid fish _Acanthochromis polyacanthus_.  Thanks to all the many
people who replied -- it generated a lot of interest!  I have summarized and
pasted relevant parts of the replies below.  For those not interested, sorry
for the long message! Again, thanks to everyone for the help. 

Kathy


----DNA degradation problems with other species----:

Butterflyfish from the Red Sea.
Squid.
Perch.
Parrotfish.

-----Suggestions for methods-----:

1) I am a graduate student working with triggerfish DNA, and I initially
thought I had encountered this same problem.  I extracted DNA from fin
clippings (however, first transferred to SED preservative) and frozen
tissue with success using a Qiagen kit, and various methods, respectively.
I am by no means an expert in this field, but I hope I can offer some
clues.  I was wondering on what basis are you determining that your DNA is
degraded?

2) This may not be your problem, but here is the situation I encountered when
 I was getting highly degraded DNA from squid.  I do extractions somewhat
similarly to what you do; the major difference may be that I do them in 1.5 ml
tubes.  There are three things that I found necessary to avoid the degradation:
(1) use very small pieces of tissue (on average about 10-15 mg and only 1 mm
thick); (2) crush them immediately in the grinding buffer (i.e., crush the
tissue from one specimen before taking tissue from the next specimen); and (3)
have the buffer already hot (tubes in the hot block at 55 C with condensation
having formed under the lid).  If I skipped any of these points, the quality of
the DNA prep would be lessened; but, it was especially critical not to use too
much tissue.  Given that you are doing phenol-type preps, you should consider
using "phase-lock gel tubes".  They do a wonderful job of sequestering the
phenol below the gel phase and the buffer above the gel... so there is no need
to avoid getting the precipitated protein transferred.  Also, I do the entire
extraction (the phenol etc. steps) in one tube and never have to pipette...
just decant.

3) what form of extraction buffer are you using i.e. does it 
contain EDTA or any chemical which will inhibit the action 
of DNAses during the digestion of the tissue.  I ask this 
because I once tried using a buffer composed of Tris, KCl 
and Tween, and found huge levels of degredation. The 
addition of EDTA largely overcame this.

4) About your DNA problem the only thing I can tell you is that I am
using the same method as you, I mean the phenol/chloroform extraction
and I tried that on tissue, gills and fin clips too. I didn't have any
big problem. It is true that with the fin clips I always had to
extract more than with tissues (4-5 times instead of 2-3 usually with
gills) but I suppose this is due a lots of tanin and proteins. Some
peole in the lab who have experienced dirt extraction and problems
with PCR afterwards use dialysis to obtain DNA and not precipitation
with ammoniac acetyl as I do (and as you maybe do) and it is true
that you obtain a very clean DNA for just one day more of time. I am
personnaly happy with the phenol extraction and ammoniac acetyl
precipitation and did not have any big problem with the Pomacentrids
I have extracted until now, except the 3 specimens I have from
Amphiprion Perideraion, but I have to try again.

5) I am working with shrimp. I have not encountered any suchproblen yet. I
use CsCl gradient for extraction of both RNA and DNA from
same sample. For DNA extraction I do proteinase K treatment followed by
Phe: Chl extraction and routinely obtain good quality of DNA.

In your case some how you seem to have DNase contamination or the
tissues you are using might have gone apoptosis. You may like to
see a recent article in Marine Molecular Biology and Biotechnology,
1996, 5($): 295-298 where they have used fins and scale of fishes to extract
DNA. Also see many cross referred articles. 

6) Degradation. ugghhh. I use fin clips and liver, but I have used the kit 
from Bio 101, and the protocol calls for a 30-120 minute incubation. I 
use less time if I think the samlple is going to be degraded (~30min). 
After following the protocol I spin down the DNA and resuspend in 25uL of 
H20. The advantage of the protocol is that it only takes about an hour 
and you can run your results on a gel right away to dtermine your amount 
of degradation. If you are trying to amplify a small area (I'm using 12S 
mtDNA) and the area if so small degradation isnt really a problem for PCR.

Also, you might try storing your fin clips in a buffer like NET* to help 
avoid degredation.

7) I noticed your request for ideas to extract DNA and remembered that we had
a similar problem with perch DNA. We solved it by increasing the lysis
buffert concentration of EDTA from 10 mM to 50 mM and increasing SDS from
0.5 to 2 %. Except for these two ingredients our lysis buffert contains
Tris-Hcl ph 8.5 100 mM and NaCl 20mM.

8)I experienced the same problem extracting DNA from dried blood, larvae and
fin clippings of parrotfish. Apparently fish have a high level of
endonuclease activity. Adding 0.5 % SDS (sodium dodecyl sulfate)to the lysis
buffer and incubation with proteinase K at 55C in stead of 37C did the trick
in my case. 
Here's the first part of my fish DNA extraction protocol:
Day 1 (late afternoon):

1 put larva or fin clip in 2,2 ml eppendorf tube, containing 1 ml TES; 0,5% SDS 

2 add 10 m l proteïnase K, mix well

3 incubate overnight at 55EC 


9)We have had similar problems with consistently obtaining
high-quality DNA from fish tissues. In particular, we have found that
while many preps will work to get DNA of sufficient quality for PCR,
mapping, etc., the DNA is not of high molecular weight for some other
more demanding protocols. In fact, we have often extracted mouse DNA in
parallel trials with the fish using such gentle methods as Recoverease,
Qiagen columns, etc. only to find the fish DNA can be highly degraded.

Fortunately, we have come up with a couple methods that result in very
good quality DNA and will be happy to pass them on. However, I  would
like more details from various researchers that have had some problems
with the fish DNA. There really does appear to be something to the
observation that "fish don't keep" and DNA degradation.

10) regarding your DNA degradation problems...my experience is that as
long as you have fragments greater than the size of your expected PCR
product, the shearing you are talking about should not be a problem.  I
like to use a 'silica matrix' or DAEA DNA extraction format (GENECLEAN or
QIAGEN).  I get less shearing because, I believe, there is less vortexing
involved.  Excessive pipetting will also result in shearing. 

11)  I'm a PhD student at the University of Tasmania, doing some molecular
phylogenetic and population genetic work on cirrhitoid fishes. You have an
interesting question. By degradation I assume that you have extracted the
DNA, treated it with RNase, run it out on a gel, and only observed low
molecular weight material. I guess you're confident of your material. Fin
clip from live fish should be good, but frozen can be highly degraded, even
if handled by protein electrophoresis people that should be careful. That
leaves the extraction technique. Your proteinase K should knock out DNases,
so that shouldn't be a problem, and some people don't prot K and get away
with it. Other potential sources of degradation.....vortexing during
phenol-chloroforms or shaking really vigorously can cause mechanical
degradation. Also, check your phenol. If oxidised, or not equilibrated
properly it could be chewing the DNA a bit. Other than that, I'm stumped.
Maybe really high DNase levels/activities? Try side-by-side DNA extraction
with material that has been extracted "sucessfully" previously, or a
different technique (ask qiagen or whoever for a free sample of their
tissue prep kit).

[that's all!]