You are on page 1of 2

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 embedded, then cut and mounted on slides. These slides were
is used extensively. Current methods for sample preparation of deparaffinized and stained, using an indirect immunofluorescence
DNA from paraffin-embedded tissues are successful, but remain assay, in order to evaluate the number of bacteria per slide.
time consuming (2, 4, 5, 6). These techniques involve two major Paraffin-embedded Coxiella burnetii-infected human cardiac valve
steps: deparaffinizing and protein digestion, each of which samples were also used. The primers used in this study have been
involves several centrifugations and washes and requires multiple described previously and are derived from the superoxide
tube transfers, which increase opportunities for the introduction dismutase gene of Coxiella burnetii (3, 7). PCR was performed
of contaminants (1). on 10 M1 of each prepared sample in a total volume of 100 /l.
We propose a one-step DNA extraction from paraffin- The final reaction mixture contained 1 $M (each) primer C.B.-1
embedded tissues direcfly suitable for PCR. This procedure and C.B.-2, 200 AM (each) dATP, dCTP, dGTP, and dTTP,
utilizes ChelexR 100 (BioRad, Richmond, CA), a chelating ion 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.0 mM MgCl2,
exchange resin. The alkalinity of ChelexR suspensions and the 0.01% gelatin, and 2.5 U Thermus aquaticus enzyme (Taq DNA
exposure to 100°C temperatures result in disruption of the cell polymerase; Promega, Madison, WI) overlaid with 100 Ml
membranes and denaturation of the DNA (8). After 30 minutes, mineral oil. Samples were subjected to 30 cycles of amplification
a DNA solution suitable for PCR is obtained. By using ChelexR (denaturation at 95°C for 20 s, primer annealing to template at
100 we were able to reduce dramatically the time of preparation 50°C for 1 min, and primer extension at 72°C for 2 min) in a
of these samples for PCR analysis and to minimize the number DNA thermal cycler (LEP Scientific, Andover, Hampshire,
of manipulation steps in order to decrease cross-contamination England). Amplification products were detected by
and contamination from extraneous DNA. electrophoresis in 2 % agarose gels, stained by ethidium bromide
Samples used concerned Coxiella burnetii, an intracellular and photographed. Uninfected L929 cells served as a negative
bacterium responsible for Q fever, a disease of human beings control.
and animals being studied in our laboratory (7). L929 cells were The procedure used here consisted of separating the sample
infected using the Nine Mile Q177 reference strain (ATCC), from the slide mechanically (using another slide or a scalpel) and
pelleted, fixed in either formalin or Bouin fixative, paraffin- mixing it (4:1) with a suspension containing 20% ChelexR 100

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
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.