Carbohydrate against DNA summary

Thomas Hiesberger hiesi at mol.univie.ac.at
Fri Nov 25 07:46:10 EST 1994


Thank you for your help and all your ideas.
Some answers have been sent to me by E-mail. I thought that the replies
will be also interesting for other people. 



The question:
_______________________________________________________________________

Hi

>I like to purify DNA out of chicken egg yolk. I always end up with a
>stuff that looks and behaves similar to DNA but is not DNA. I think it is
>a carbohydrate. I wonder if somebody could give me an idea how to remove
>this substance. 
>Now more exactly: I dilute the yolk and make an over night Protein
>degradation with proteinase K. Then I perform a Phenol/Chloroform
>extraction. After the precipitation with isopropanol I get this white
>huge pellet which is not DNA. This Substance cannot be removed with
>Phenol, not digested with amylase or with hyaluronidase. It is water
>soluble (better soluble in acidic solution), makes a kind of gel in basic
>solution. And makes me nervous. What is it? How to remove it. I would be
>grateful for any help
>
>Thanks
>Thomas Hiesberger
>Institute for molecular genetics
>University Vienna
_______________________________________________________________________


The answers:

1. 
Even if you remove the mystery glop, one non-fertilized egg (=one cell) 
should have only about a pg of DNA in it.  What are you planning to do 
with it?  If you just need chicken DNA, why not consider blood?  Red 
cells are app. 5 exp9 cells/ml, and are nucleated in birds, so one drop 
(say 50 ul) should have several ug of DNA.

If you need the ovum DNA, I guess you should probably throw away the yolk 
and try to find the nucleus, if possible,  I'm not a developmental 
biologist, so I can't tell you how to do this, though.
Nick


2. 

	Unfortunately I do not work with chickens but instead I work with trees
.......anyway when I extract DNA from conifers many times starch and
carbohydrates
 are associated with the DNA.  The best way to remove them I find is to
perform 
a spermine ppte.
The proceedure that I use is based on a method published in Nucleic Acids
Research
volume #9(20)(1981) by Barbara Hoopes et al. pages 5493-5504 called
"Studies on 
the Selectivity
of DNA preciptitation by Spermine".  After the isopropanol ppte.
resuspend your 
DNA in a suitable
volume of TE and then follow the directions in the paper. I have also
used 
another method to remove
unwanted junk from my DNA; a method called differential salt ppte. 
Basically 
you ppte. your
DNA in high salt conc. followed by a second ppte in lower salt conc. 
However 
this method never
seemed to work for me. Getting back to the spermine ppte. other people
doing 
work in flies say 
that a spremine ppte.is the best way to remove any kind of junk from DNA 
regardless of organism.
Anyway feel free to e-mail me if you have any questions about this stuff.
Cheers,
Mark Hicks

3. 
You could use Cetylmethylammonium bromide from Sigma #5882,
which you can see in one article of M.G.Murray and W.F.Thompson
volume 8 number 19 1980 Nucleic Acids Research how to do.
You also should check Methods in Enzymolozy vol. 152
Silva

4. 
Dear Thomas -
2 thoughts on your problem:
My first reaction is that the white substance could be NaCl, which
coprecipitates w/DNA in isopropanol.  Try using ethanol and see if it
disappears.  (Just an idea - I can't explain why it is more soluble in
acidic solution).

Secondly, have you considered a CTAB (hexadecyltrimethylammonium bromide)
extraction?  This is good at getting rid of polysaccharides.  After
proteinase K digestion,
1.  bring the [NaCl] to 0.7 M
2.  add 0.1 volumes of (10% CTAB in 0.7 M NaCl).  Heat at 65 degrees C for
10 min
3.  CHCl3/IAA extract
4.  phenol/CHCl3/IAA extract
5.  ppt the DNA - note the high [NaCl], so you'll need to do a couple 70%
EtOH washes.

Good luck
Robert Schoenfeld

5. 
	The lipids in the egg do tend to give funny reverse phases.   You 
might want to try and extract the lipids out of your system with
chloroform
twice before using isopropanol.  I hope it works.  Good Luck!!!!!

	Amrut

6. 

There is so little DNA in an egg yolk that I doubt you will ever be 
successful. An egg contains a single diploid or tetraploid nucleus, 
so even with 100% recovery you would end up with a few picograms of DNA 
per egg. In any case, what you describe sounds like polysaccaride, many 
of which co-purify with DNA, and are not removed by any of the 
techniques you describe. I don't know how to get rid of polysaccaride; 
some polysaccaride contamination is not usually a problem, but then most 
applications don't have your problem that you are trying to recover 
picograms of DNA from an enormous mass of tissue.

Ed Stephenson

7. 


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