Acid-phenol extraction of circular DNA

Bill Melchior, NCTR/FDA wmelchior at NTBTOX.NCTR.FDA.GOV
Fri Jun 19 13:46:36 EST 1992


>Does anyone have any experience with the purification of closed
>circular DNA by extraction from low-ionic-strength, low-pH buffer,
>using phenol equilibrated to pH 4.0 with NaOAc?  This method was
>published by Zasloff et al. '78 (NAR 5:1139-1152), and it looks both
>easier and higher-yield than ethidium bromide-CsCl centrifugation, but
>it doesn't seem to be in common use.  The claim is that, for reasons
>"not as yet understood" (any ideas?), linear and nicked circular DNA
>partition into the organic phase under these conditions, while
>covalently closed rings remain in the aq. phase.  The method is good
...
>What I'm trying to do is to isolate and clone extrachromosomal closed
>circular DNA (eccDNA, aka cccDNA, aka spcDNA) from mammalian cells
>growing in vitro.  The above method, originally developed for plasmid
>preps, has in fact been used for this purpose [Truett et al. '81 Cell
>24:753-763], but it doesn't seem to have caught on, so I'd appreciate
>hearing from anybody who has actually tried it, either for plasmids or
>for organelle, viral, or eccDNA.
>
>I need high purity, because I want to be able to say with confidence
>that any resulting clone came from extrachromosomal DNA & not from
>contaminating chromosomal fragments.  (There is an ATP-dependent DNAse
>method by Yamagishi et al '83 [Gene 26:317-321], but it only gives 96%
>purity.)  And the high yield of the acid-phenol method is very
>attractive, because my starting material is available in limited
>quantity (10^9 cells max).  I have heard that the yield after 3
>successive CsCl runs (which is what I would need for the requisite
>purity, & seems to be the standard method in the literature) is as low
>as 10%.  Any feedback on whether that's true?  I also have concerns
...

Anita-

I've not used the Zasloff procedure, but until I started using a glassmilk
procedure for plasmid purification, my best sequences were with an acid
phenol method: Weickert and Chambliss, USB Comments v16 #2, 5-6 (summer
1989).  Extraction here is from a HIGH ionic strength buffer; the acid phenol
(pH 4 against NaOAc) is used after an SDS/NaOH lysis and a 3M KOAc precip-
tation of unwanted macromolecules.  My biggest problem with it was that the
yield of plasmid was lower than by other methods, even when I decreased the
volume of phenol to 1/2 of the amount called for.  (DNA is more soluble in
acid phenol, at least under these circumstances.)

The resulting product usually showed NO bacterial DNA, but: (1) Occassionally
I could see a probable hint of some on a gel (very subjective call).
(2)  Nothing visible on a gel is probably not a sufficiently stringent
test to meet your requirements; a few percent contamination would not be
visible unless I really overloaded the plasmid on the gel.  

As far as CsCl/ethidium bromide gradients, I've never quantitated my yields, 
but suspect that it's generally better than 80% from a single run, which would
translate to >50% for three runs.  I am, however, doing pBR322 from 800 ml 
cultures of coli; I suspect that your worries about quantities from euk cells 
are justified.  (Three cheers for bacteria.  I know many people who work with
animals and/or eukaryotic cells, and I'm am always glad that I don't have to 
deal with their problems.)

(PS, I collect DNA from the top of the centrifuge tube, using a siliconized
capillary tube on a Micro Pipex controller.  I get great control over the
process this way, and can stop whenever I need to, collect multiple bands
without difficulty, etc.)

I hope you get some replies from someone who's worked with eukaryotic cells.
Good luck, Bill
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators



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