Nucleic Acids Research, Vol. 20, No.
19 5237-5238
A simple method for amplification of DNA from paraffin-
embedded tissues
Andreas Stein and Didier Raoult*
Unite des Rickettsies, Faculte de Medecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05,
France
Submitted August 17, 1992
PCR examination of DNA from fixed paraffin-embedded tissues
is used extensively. Current methods for sample preparation of
DNA from paraffin-embedded tissues are successful, but remain
time consuming (2, 4, 5, 6). These techniques involve two major
steps: deparaffinizing and protein digestion, each of which
involves several centrifugations and washes and requires multiple
tube transfers, which increase opportunities for the introduction
of contaminants (1).
We propose a one-step DNA extraction from paraffin-
embedded tissues direcfly suitable for PCR. This procedure
utilizes ChelexR 100 (BioRad, Richmond, CA), a chelating ion
exchange resin. The alkalinity of ChelexR suspensions and the
exposure to 100°C temperatures result in disruption of the cell
membranes and denaturation of the DNA (8). After 30 minutes,
a DNA solution suitable for PCR is obtained. By using ChelexR
100 we were able to reduce dramatically the time of preparation
of these samples for PCR analysis and to minimize the number
of manipulation steps in order to decrease cross-contamination
and contamination from extraneous DNA.
Samples used concerned Coxiella burnetii, an intracellular
bacterium responsible for Q fever, a disease of human beings
and animals being studied in our laboratory (7). L929 cells were
infected using the Nine Mile Q177 reference strain (ATCC),
pelleted, fixed in either formalin or Bouin fixative, paraffin-
257-
Figure 1. 257-bp amplification product of DNA from 8 paraffin-embedded tissue samples containing C.bumetii microorganisms. Lanes B-E fixation in formalin,
Lanes G-J fixation in Bouin fixative, Lanes A,F,K,M: molecular size markers (+X174 cleaved with HaellI) Lane L: reagent control (uninfected L-929 cells).
* To whom correspondence should be addressed
embedded, then cut and mounted on slides. These slides were
deparaffinized and stained, using an indirect immunofluorescence
assay, in order to evaluate the number of bacteria per slide.
Paraffin-embedded Coxiella burnetii-infected human cardiac valve
samples were also used. The primers used in this study have been
described previously and are derived from the superoxide
dismutase gene of Coxiella burnetii (3, 7). PCR was performed
on 10 M1 of each prepared sample in a total volume of 100 /l.
The final reaction mixture contained 1 $M (each) primer C.B.-1
and C.B.-2, 200 AM (each) dATP, dCTP, dGTP, and dTTP,
50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.0 mM MgCl2,
0.01% gelatin, and 2.5 U Thermus aquaticus enzyme (Taq DNA
polymerase; Promega, Madison, WI) overlaid with 100 Ml
mineral oil. Samples were subjected to 30 cycles of amplification
(denaturation at 95°C for 20 s, primer annealing to template at
50°C for 1 min, and primer extension at 72°C for 2 min) in a
DNA thermal cycler (LEP Scientific, Andover, Hampshire,
England). Amplification products were detected by
electrophoresis in 2 % agarose gels, stained by ethidium bromide
and photographed. Uninfected L929 cells served as a negative
control.
The procedure used here consisted of separating the sample
from the slide mechanically (using another slide or a scalpel) and
mixing it (4:1) with a suspension containing 20% ChelexR 100
5238 Nucleic Acids Research, Vol. 20, No. 19
(Bio-RAD, Richmond, VA) in a 0.1% Lauryl sulfate, 1%
Nonidet P40, 1 % Tween 20 aqueous solution. The mixture was
boiled for 10 min, the paraffin then appeared floating on the
surface of the solution. The samples were centrifuged (12000
g for 10 min) and the supernatant was used directly for PCR
analysis. Using these conditions 8 samples were studied from
cells fixed with either formalin or Bouin fixative (Figure 1). The
same slides stained by indirect immunofluorescence showed 102
to 103 organisms.
Paraffin-embedded human heart valve samples from patients
in which Coxiella bumetii had been isolated were treated the same
way and were successfully amplified.
When we have subjected samples (both embedded cells and
embedded heart valve tissues) to simple boiling treatment in
distilled water alone, the DNA was not detected by PCR.
In summary, this procedure should permit the rapid preparation
of DNA for PCR from paraffin-embedded samples. Since fewer
manipulations are required, it should also reduce the chance of
inadvertent contamination of samples by extraneous DNAs. We
believe that this new application of ChelexR 100 chelating resin
will be helpful for routine treatment of paraffin-embedded
samples.

ACKNOWLEDGEMENT
The authors thank X. De Lamballerie for helpful discussions.

REFERENCES
1. Erlich,H.A. (1989) PCR Technology. Principles and applications for DNA
amplification. Stockton Press, New York.
2. Goelz,S.E., et al. (1985) Biochem. Biophys. Res. Commnun. 130, 118- 126.
3. Heinzen,R.A., et al. (1990) Nucleic Acids Res. 18, 6437.
4. Impraim,C.C., et al. (1987) Biochem. Biophys. Res. Commun. 142,
710-716.
5. Shibata,D.K., et al. (1988) J. Exp. Med. 167, 225-230.
6. Shibata,D.K., et al. (1988) Cancer Res. 48, 4564-4566.
7. Stein,A. and Raoult,D. (1992) J. Clin. Microbiol. 30, 2462-2466.
8. Walsh,P.S., et al. (1991) BioTechniques 10, 506-513.