Hot/Acid Phenol Extraction

Michael G. Tencza tencza at med.unc.edu
Tue Dec 28 12:32:56 EST 1993


   Hi Netters and Happy Holidays!

   Sorry about the last bogus post, but I hit the wrong key somewhere.
   I would like to see if anyone could clear up the following
   controversy.  Recently I became interested in RNA extractions and
   ran across a method using phenol pH balanced to 5.2 with sodiom
   acetate and incubating the sample/phenol mixture in a 55C water
   bath for 3-4 minutes prior to centrifugation.  I querried several
   sources about this procedure and obtained conflicting information.
   One source told me that the acid pH of the phenol causes DNA to end
   up in the organic phase and selectively leave the RNA in the
   aqueous phase, while another said that both RNA and DNA end up in
   the aqueous phase no matter the pH of the phenol).  If anyone has
   experience with this method I would appreciate a response to shed
   light on the selectivity of this method.  Thanking you in
   advance...

                                           Mike

I have a couple of references on a slightly different use of acidic
phenol, namely separation of covalently closed circular DNA from
nicked & linear.  Unfortunately they don't seem to discuss RNA, but
they do discuss which DNA species partition into which phase at
various pH's & salt concentrations.  It does seem pretty clear that
the non-cccDNA ends up in the organic phase.  Azad et al (referencing
Muller) says that the non-cccDNA is denatured by both the phenol & the
low pH, & that the resulting ssDNA is hydrophobic enough to partition
into the phenol (given charge-compensation of the PO4's by appropriate
Mg++ concentrations).  (I guess the cccDNA is protected from this fate
by its favorable renaturation kinetics.)

I'll leave it to you to figure out what happens with RNA, but maybe
your method above is delicately balanced to melt dsDNA but not disrupt
RNA secondary structure too much.

Here are the refs:

M Zasloff et al, A new method for the purification & identification of
covalently closed circular DNA molecules.  NAR 5:1139-1152 (1978).

AK Azad et al, An improved method for rapid purification of covalently
closed circular plasmid DNA over a wide size range.  Lett Applied
Microbio 14:250-4 (1992).

I don't have the following paper, but it looks like what you're really
after:

D. Muller et al, Aspects of the mechanism of acid-phenol extraction of
nucleic acids.  Biochim Biophys Acta 740:1-7.

Hope this helps.  Good luck!

                                   -Anita Gould
                                    anita at cco.caltech.edu



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