DNA extraction from formalin-fixed material

[John Compton]

In article <fodde.8.00089130 at ruly46>, fodde at ruly46 (Riccardo Fodde) wrote:

> Hi everyone, we have got a small technical problem: we have received some 
> tumor samples fixed in formalin and subsequently stored in 70% EtOH (N.B. they 
> have NOT been embedded in paraffin). We would like to isolate DNA from these 
> samples. Does anyone have a good protocol?
> 

I requested help with this same problem earlier this year.  I append the
response from Georgia Trench.  The referenced method has worked well in my
hands...and is very simple and rapid.  Some samples may prove negative,
however.  You should then try a second section.  Also, 40 PCR cycles may
prove useful in some cases (small tissue samples), or a reamplification of
the initial reaction may yield less artifact bands.  

*We have had great success with the very simple method by Levi et al
(Cancer
*Res 51: 3497-3502, 1991) in which you just incubate sections with
proteinase
*K and Tween 20 (2-12 hours on rocking platform at 55C) and then boil
before
*using in PCR.  We sometimes follow this with Instagene clean up.  Although
I
*have heard that different fixation methods can give problems this method
*almost always works for us (albeit never as good as a pure DNA prep - for
*example it is very difficult to multiplex with it).  My feeling from
talking
*to others is that most of the problem with PCR from fixed tissue comes
from
*an excess of organic extractions so that there is no DNA left.  Levi's
*method is dead simple and quick, and avoids this.  Hope this is useful. 
*                Georgia Trench

Good luck!! 
-- 
John G. Compton
Lab of Skin Biology
NIAMS, NIH Building 6 room 425
Bethesda, MD.
jcomp at helix.nih.gov
301-496-7193
-- 
John G. Compton
Lab of Skin Biology
NIAMS, NIH Building 6 room 425
Bethesda, MD.
jcomp at helix.nih.gov
301-496-7193