Yonago Acta medica 1996;39:171–176

Improvements in the Detection of Epstein-Barr Virus DNA on ParaffinEmbedded Gastric Carcinoma Tissues: Treatment of Extracted
Cellular DNA with a Restriction Enzyme Prior to Polymerase Chain
Reaction
Noriko Takasaka, Yukio Satoh, Yoshiko Hoshikawa, Mitsuhiko Osaki*, Hisao
Ito*, Jen-Yang Chen† and Takeshi Sairenji
Department of Biosignaling and *First Department of Pathology, Faculty of Medicine, Tottori
University, Yonago 683 and †Department of Bacteriology and Graduate Institute ofMicrobiology, College of Medicine, National Taiwan University, Taipei, Taiwan
An association between Epstein-Barr virus (EBV) and a certain gastric carcinoma has
been suggested by the presence of EBV-DNA in the carcinoma cells. We tried to detect
EBV-DNA in paraffin-embedded tissues of gastric carcinoma by polymerase chain reaction (PCR) followed by Southern analysis. The EBV-DNA sequence was not amplified
sufficiently with a BamH I-W primer pair to the extracted DNA itself from the tissues
which were detected for the expression of EBV-encoded small RNA (EBER), while thesequence was amplified to the DNA from Raji cells as a positive control. However,when the extracted DNA was digested with a restriction enzyme, BamH I or EcoR I andthen subjected to PCR, the BamH I-W region was amplified in the DNA from EBERpositive but not -negative tissues. In the extracted DNA from an EBV genome-carrying
cell line derived from a gastric carcinoma, the amplification with PCR was increasedslightly by the digestion with BamH I. Thus, the pretreatment for template DNA
increased the sensitivity of PCR for the detection of EBV-DNA from paraffin-embeddedtissues.
Key words: Epstein-Barr virus; gastric carcinoma; PCR; restriction enzymeal., 1988; Saito et al., 1989; Sallie et al., 1992).
It has been reported that the EBV genome has
been detected in DNA extracted from paraffinembedded tissues by PCR (Shibata et al., 1991;Min et al., 1991; Fukayama et al., 1994; Imai etal., 1994). There are reports which describe theimprovement of PCR detection from paraffinembedded tissues (Kiene et al., 1992; Sepp etal., 1994). We tried to detect the EBV genome
more effectively in DNA extracted fromparaffin-embedded gastric carcinoma tissues byPCR-Southern analysis and found that the detection of EBV-DNA was improved by the pretreatment of the restriction enzyme for extractedDNA.

In recent studies, the Epstein-Barr virus (EBV)
genome was detected in about 7% of gastric carcinomas in Japan and was detected in carcinoma
cells of most cases of gastric carcinoma withlymphoid stroma (Shibata et al., 1991; Min et al.,
1991; Tokunaga et al., 1993; Fukayama et al.,
1994; Imai et al., 1994). These results suggestthat EBV might be associated with the pathogenesis of some gastric carcinomas. It is a significant issue to detect the EBV genome in carcinoma tissues with high sensitivity and specificity for the diagnosis of EBV-related gastric carcinoma. The use of polymerase chain reaction(PCR) for rapid diagnosis of infectious agentshas been reported in several studies (Shibata etAbbreviations: bp, base pair; EBER, EBV-encoded small RNA; EBV, Epstein-Barr virus; PCR, polymerase
chain reaction

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N. Takasaka et al.

PCR-Southern analysis

Materials and Methods

Specimens
The tissues were from surgical specimens of gastrectomy. Formalin-fixed paraffin-embeddedtissues from gastric carcinoma with lymphoidstroma were tested for the expression of EBVencoded small RNA (EBER; Glickman et al.,1988) as previously described (Tokunaga et al.,1993), and used for the detection of EBV-DNAby PCR.

Cells
Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37˚C in a
CO2 incubator. Epithelial cells were passaged
with 0.25% trypsin and 0.02% EDTA. EBV
genome-carrying Raji cells (Pulvertaft, 1965)
and the genome-free Ramos cells (Klein et al.,1975) were used as positive and negative
controls respectively for PCR analysis.

DNA extraction
Twenty pieces of a 10 µm section were cut fromthe paraffin blocks and transferred into sterile
15 mL plastic tubes. Paraffin was removed bytreatment with xylene. The sections werehydrated with a graded alcohol series of 100%,70% and 50% ethanol and deproteinated with
200 µg/mL proteinase K at 37˚C overnight.
The DNA was purified by phenol-chloroform
extraction and ethanol precipitation and dissolved in 1 mL TE (10 mM Tris-HCl, pH 8.0, 1mM EDTA).
For DNA extraction from cultured cells, 106
cells were washed with 10 mM phosphatebuffered saline, pelletted, resuspended in distilled water and lysed by 2 cycles of freezingthawing. The DNA was extracted by 50 mM
NaOH at 95˚C for 10 min, neutralized with 1 M
Tris-HCl (pH 7.5) and purified by phenolchloroform extraction and ethanol precipitation. It was then dissolved in 50 µL TE.

The DNA (1 µg) in 5 µL TE was diluted into 50µL of a solution containing 200 µM each of
deoxyribonucleoside triphosphates (dNTPs), 2mM MgCl 2 , 500 nM each primer, 2.5 U Taq
DNA polymerase (Takara Shuzo Co., Ltd.,
Kyoto, Japan). The primers were designed toamplify a 129-base pair (bp) segment in theBamH I-W region (5'-CCAGACAGCAGCC
AATTGTC-3' and 5'-GGTAGAAGACCCCCTCTTAC-3') of the EBV genome as previously described (Tomita et al., 1995). PCR wasperformed in one cycle for 3 min at 94˚C, 1 minat 58˚C and 1 min at 72˚C, and in 34 cycles for 1min at 94˚C, 58˚C and 72˚C, respectively. The
amplified products were electrophoresed in 2%
agarose gel and transferred to a Hybond N +membrane (Amersham Japan Co., Ltd., Tokyo,Japan). A 40-base oligonucleotide probe (5'CCCTGGTATAAAGTGGTCCTGCAGCTATTTCTGGTCGCATC-3') (Uhara et al., 1990),
which hybridized to intervening sequence between 2 EBV primers was 5'end-labeled with a[γ-32P]ATP. The membrane was hybridized
with the labeled probe at 42˚C overnight andFig. 1. Detection of BamH I-W region in the EBVgenome on Raji cells by PCR-Southern analysis.
The serial 10-fold diluted cellular DNA of Raji (105cells or 5 × 10 6 copies of viral genomes) weresubjected to PCR (lane 1, 5 × 10 6 copies of EBVgenome; lane 2, 5 × 10 5 copies; lane 3, 5 × 10 4copies; lane 4, 5 × 103 copies; lane 5, 5 × 102 copies;
lane 6, 5 × 101 copies; lane 7, 5 × 100 copies; lane 8,5 × 10 –1 copies). Ramos cells were used as thenegative control (lane 9). The same amount (5 µL)
of PCR reaction mixture was electrophoresed.

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washed in washing buffer (2 × SSC, 0.1% SDS)at 50˚C. Autoradiography was carried out at–80˚C with X-ray film.

showed that the PCR had high sensitivity enoughto detect even one copy in the template DNA.

BamH I or EcoR I digestion

Detection of EBV-DNA in paraffinembedded tissues of gastric carcinoma
by PCRThe DNA (1.8 µg) extracted from the paraffinblocks were dissolved in 10 µL of buffer K (20mM Tris-HCl, pH 8.5, 10 mM MgCl 2, 1 mM
DTT, 100 mM KCl) or buffer H (50 mM TrisHCl, pH 7.5, 10 mM MgCl 2 , 1mM DTT, 100mM NaCl) containing 10 U BamH I or EcoR I(Takara Shuzo Co.), and incubated at 30˚C or37˚C, respectively for 1 h. The reaction mixtures were incubated at 60˚C for 15 min to inactivate the enzyme, and the DNA was ethanolprecipitated and dissolved in 5 µL TE.

The PCR-Southern analysis was carried out todetect the EBV genome in the DNA fromparaffin-embedded tissues of gastric carcinoma(Fig. 2A). However the BamH I-W region was
not amplified to a detectable level to the extracted DNA by PCR in EBER-positive (lanes 1–3)
or -negative tissues (lanes 4–7), while the region was amplified in DNA from the EBV
genome-positive Raji cells (lane 9). In order tofind the problem for no amplification, anextracted DNA from an EBER-positive tissue
was pretreated by several methods prior to PCR(Fig. 2B). The treatments were as follows: i)the shearing of DNA through a needle, ii)
digestion with BamH I, iii) boiling with analkali solution, and iv) the addition of Raji
DNA to test the presence of any inhibitor(s) toPCR. The amplified EBV-DNA band wasdetected in BamH I digestion (lane 3), alkalitreatment (lane 4) or the DNA mixture with RajiDNA (lane 5) but not in the shearing of DNA(lane 2). The amplification of DNA in themixture with Raji DNA indicated that there wasno inhibitor for PCR in the extracted DNA.
DNA samples which were not detected for
EBV-DNA by PCR in Fig. 2A were digested
with BamH I and then subjected to PCR (Fig.
2C). The band to BamH I-W was detected in all
DNAs from EBER-positive (lanes 1–3) but notin -negative tissues (lanes 4 –7).
The effect of another restriction enzyme,EcoR I was examined for DNA extracted froman EBER-positive tissue (Fig. 2D). The BamH
I-W region was also amplified similarly.

Shearing or alkali treatment of DNA
The DNA in TE was sheared mechanically bypassing through a 26 gauge needle 10 times.
Part of the DNA sample (1.8 µg) in 5 µL TE
was then mixed with 50 µL 50 mM NaOH,
layered by 50 µL mineral oil, heated at 95˚C for
10 min and neutralized by the addition of 8 µL
1 M Tris-HCl, pH 7.5. After removal of themineral oil by treatment with chloroform, theDNA was precipitated with ethanol and dissolved in 5 µL TE.

Results

Sensitivity of PCR
The sensitivity of PCR-Southern analysis todetect EBV-DNA was tested in serially 10-folddiluted template DNA obtained from Raji cells
that have 50 copies of EBV-DNA per cell (zurHausen et al., 1972) (Fig. 1). A band at 129 bp
was detected in all the PCR of the diluted template DNA (lanes 1– 8), with BamH I-W probe,
but not in the PCR of DNA from EBV genomefree Ramos cells. The PCR amplification
could detect 5 × 10–1 copies of EBV genome(lane 8) which was estimated from the copies ofviral genome in diluted Raji DNA. This result

Effect of BamH I digestion of DNA extracted from a cultured EBV positive cellline derived from a gastric carcinoma

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DNA was extracted from an EBV-positive cellline (Yanoma et al., unpublished data) derived

N. Takasaka et al.

Fig. 2. PCR-Southern blot analysis of amplified DNA with BamH I-W primer. (A) Detection of EBV
genomes in gastric carcinoma with lymphoid stroma. The DNA extracted from paraffin-emdedded tissues
was amplified by PCR and detected with Southern blot. (lanes 1–3, EBER positive tissues; lanes 4–7, EBER
negative tissues; lane 8, Ramos; lane 9, Raji). (B) PCR amplification of DNA pretreated by several
procedures as described in Materials and Methods. (lane 1, Raji DNA; lane 2, shearing; lane 3, BamH I
digestion; lane 4, treated with alkali solution; lane 5, Raji DNA (the same as lane 1) plus DNA extracted froma paraffin block (the same as lane 2); lane 6, Ramos DNA). (C) Effect of BamH I digestion on PCR. The
extracted DNA was digested with BamH I and analyzed by PCR-Southern blot. (lanes 1–3, EBER positive;lanes 4 –7, EBER negative; lane 8, Raji; lane 9, Ramos). (D) Effect of EcoR I digestion for detection of EBVDNA in PCR. The extracted DNA from an EBER-positive tissue was digested with EcoR I and amplified
with BamH I-W primer by PCR. (lane 1, Raji; lane 2, no digestion; lane 3, BamH I digestion; lane 4, nodigestion; lane 5, EcoR I digestion; lane 6, Ramos)

Fig. 3. Effect of BamH I digestion on DNA from cultured EBVgenome-positive cells. DNA was extracted from an EBV-positive cellline derived from a gastric carcinoma and digested with BamH I andanalyzed by PCR-Southern analysis. (lane 1, no digestion; lanes 2–3,10- and 100-fold dilutions of PCR product of lane 1; lane 4, BamH I
digestion; lanes 5–6, 10- and 100-fold dilution of PCR products oflane 4; lane 7 Raji).

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Detection of EBV DNA by PCR

been used more frequently for diagnosis of
EBV infection for paraffin-embedded tissues
from several malignant diseases (Tomita et al.,
1995; Van Rensburg et al., 1995; Vasef et al.,1995). The treatment of a restriction enzyme
will be useful in improving the detection ofEBV-DNA for such materials.

from a gastric carcinoma and digested withBamH I and analyzed with PCR. The amplification for BamH I-W was increased slightly indigested DNA than non-digested DNA (Fig. 3).

Discussion
In the present study, we demonstrated that digestion of extracted DNA with restriction enzymes, BamH I or EcoR I was a useful procedure to detect the EBV genome for DNA extracted from paraffin-embedded tissues byPCR. Restriction enzymes, BstX I, Cla I, Kpn I
or Sal I, which have no cleavage site(s) in theBamH I-W fragment, were similarly effective(data not shown). We do not know the reason
why the amplification of PCR is increased by
the digestion with a restriction enzyme; however, these results may indicate that PCR reactivity could be increased by the exposure ofthe BamH I-W region to the fragmentation ofcellular DNA. Taq polymerase containing theassociated 5' to 3' exonuclease activity facilitates a nick translational DNA synthesis (Longleyet al., 1990). If the DNA from paraffin-embedded
tissues has many nicks, then the nicks could
serve as primers for the DNA synthesis. Shortening of DNA by digestion will result in quicker
release of the polymerase from DNA and amore frequent reassociation to the next primer
for DNA amplification.
We observed no amplification of EBVDNA in DNA from paraffin-embedded gastriccarcinoma tissues without the pretreatment ofrestriction enzyme, although the sensitivity of
PCR was very high to detect EBV-DNA for
EBV-positive Raji cells. A possibility may
depend on the configuration of EBV-DNA ingastric carcinoma cells. The configuration ofEBV-DNA has not been known in cells ofgastric carcinoma. EBV-DNA may exist in adifferent mode, such as the integration in cellular DNA which differs from the episomal
DNA as seen in most EBV-positive B cell lines.
PCR amplification may be increased by the digestion of cellular DNA with a restriction enzyme
which exposes the integrated EBV-DNA.
Detection of the EBV genome by PCR has175

Acknowledgments: This work was supported by aGrant-in-Aid for the 2nd Term Comprehensive 10Year Strategy for Cancer Control from the Ministryof Health and Welfare of Japan. We thank Dr.
Shunsuke Yanoma for providing an EBV-positive
gastric carcinoma-derived cell line.

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(Received September 2, Accepted September 30, 1996)

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