Journal of Virological Methoris, 36 (1992) 63-12 63

0 1992 Elsevier Science Publishers B.V. / All rights reserved / Ol66-0934/92/%03.50

VIRMET 01272

A simple and rapid method of high quantity DNA

isolation from cervical scrapes for detection of
human papillomavirus infection

V. Gopalkrishna, Adeline Francis, J.K. Sharma and B.C. Das

Division of Molecular Oncology, Institute I$ Cytology and Preventive Oncology (ICMR),
New Delhi, India

(Accepted 4 September 1991)

Summary

Infection with human papillomavirus (HPV) is an important etiological

factor in the development of cervical cancer, and detection of the viral genome
is of prognostic importance, particularly for preneoplastic lesions. We
developed a simple, easy and efficient non-organic method of DNA extraction
from cervical scrapes for reliable detection of HPV DNA sequences. The
method involves incubation of cell nuclei in higher concentration of proteinase
K at 65°C for 2.5 h. Following prolonged incubation at higher temperature, the
enzyme is autoinactivated and the DNA isolated can be used directly for
analysis without further purification. The recovery of DNA is more than 95%
and it can be easily cleaved by restriction enzymes and is suitable for
amplification by the polymerase chain reaction (PCR). The whole procedure is
carried out in a single Eppendorf tube and a large number of specimens can be
processed at a time without any error of handling. DNA extracted from a single
smear sample is sufficient to conduct as many as four different molecular
biology tests. This provides an opportunity for verification of sensitivity,
specificity and reliability of each test for diagnosis of HPV infection without
resorting to biopsy.

DNA extraction; Proteinase K; Human papillomavirus; Southern blotting; Filter in

situ hybridization; Polymerase chain reaction

Correspondence to: B.C. Das, Division of Molecular Oncology, Institute of Cytology and Preventive
Oncology (ICMR), Maulana Azad Medical College Campus, New Delhi I10 002, India.
64

Introduction

The association of human papillomavirus (HPV) infection with cervical

carcinogenesis is well documented. The influence of certain distinct HPV types
as ‘high risk’ types (HPV 16 and 18) in the clinical course of the disease and on
progression of the lesion has also been demonstrated (Gissman et al., 1987; zur
Hausen, 1989; Das et al., 1989). HPV typing is therefore of both diagnostic and
prognostic importance. In the absence of serological tests, HPV typing is
exclusively by nucleic acid hybridization techniques using cellular DNA
isolated from tumour biopsy specimens. As biopsies are not available from
preneoplastic lesions and as the yield of DNA from cervical scrapes of usually
small cervical dysplasias is often inadequate, a technique of filter-in-situ
hybridization has been recently introduced where cells from cervical swabs/
scrapes are directly filtered onto nitrocellulose membrane, denatured and
hybridized to detect HPV infection (Wagner et al., 1984; Wickenden et al.,
1985). Filter-in-situ hybridization (FISH) allows screening of a large
population but is a cumbersome method and has limitations in interpreting
the autoradiograms. This is because of its low sensitivity and/or specificity.
There are also problems of higher background due to obvious contamination
of mucous and blood in cervical scrapes. In addition, the results of FISH
cannot be compared with those achieved by other methods since no additional
tests can be carried out due to lack of adequate material. Therefore extraction
of a sufficient amount of DNA from scraped cells is essential for performing
dot/slot or Southern blot hybridization or polymerase chain reaction (PCR)
which are more sensitive and reliable methods.
The conventional method of DNA extraction involves lysis in SDS/sarkosyl
and digestion with proteinase K followed by extraction with phenol and
chloroform plus iso-amylalcohol and subsequent precipitation in absolute
ethanol. Here we describe a simple two-step method which eliminates the need
for hazardous organic chemicals, such as phenol and chloroform, for an
efficient extraction of DNA from cervical scrapes. Since the yield of DNA is
very high by this method as compared to that of the conventional phenol-
chloroform method it offers a unique opportunity to conduct additional
experiments as well as to compare and verify the results obtained by FISH with
that of dot/Southern blot hybridization or PCR on the same specimen.

Materials and Methods

Patients and specimens

Cervical scrapes from ten women visiting the Gynaecological OPD clinic at
Lok Nayak Jaiprakash Narayan Hospital, New Delhi, with different grades of
dysplasia and cancer, were collected in 5 ml PBS (phosphate-buffered saline)
and stored at -70°C until analysis. Scrapes were taken from each patient by
 65

scraping the ectocervix or the surface of the portio with a wooden spatula.
After preparing a smear on a slide for the conventional PAP staining required
for cytopathological diagnosis, the remaining materials, along with the spatula,
were transferred to bottles containing PBS.
The adequacy of the materials was confirmed by counting the cells before
dividing into individual tests. All specimens contained more than 1-3 x lo5
cells. After vortexing, only 2 ml was processed for DNA extraction while the
remaining 3 ml was used for FISH.

DNA extraction

The method involves mainly two steps: washing in Tris-Triton buffer (TTB)
and proteinase K digestion in Tris-EDTA buffer (TEB).
2 ml PBS solution containing scraped cervical cells were taken in a 2.2-ml.
Eppendorf tube and microfuged for 2 min. The cell pellet was washed once in 1
ml cold PBS and twice in 1 ml chilled TTB containing 10 mM Tris-HCl (pH
S.O), 10 mM MgC12, 300 mM sucrose and 0.8% Triton X100. The pellet was
collected by microfuging for 2 min. The pellet was rewashed in 0.5 ml cold TE
buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA and 10 mM NaCI.
Additional washings were given if the detergent used was not removed
completely. Finally the pellet was resuspended in 200 ul TE buffer
supplemented with 1.25 mg/ml proteinase K (Boehringer Mannheim, Cat.
No. P.161519, Germany) and incubated at 65°C water bath for 2.5 h. The tubes
were vigorously shaken every 15-20 min to allow uniform lysis of the pellet and
efficient extraction of DNA. When the solution became transparent it was
assumed that the extraction was completed. The whole procedure was carried
out in a single Eppendorf tube and took only 3 h. Following determination of
the DNA concentration by gel electrophoresis (see Fig. 1) the DNA was
directly used for restriction digestion and Southern blotting. This DNA if
required, could be processed for further purification by the phenol-chloroform
method or precipitated by adding an equal volume (200 ~1) of distilled water, l/
10 vol. of chilled 3 M sodium acetate (pH 5.0) and 2.5 vol. of absolute ethanol.

Probe preparation and radiolabelling

Since HPV types 6 or 11 and 16 or 18 are predominantly detected in

preneoplastic and neoplastic lesions, respectively (see de Villiers, 1989),
recombinant plasmids containing the above HPV types were used as probes
in the present study. Gel separated and DEAE membrane (Serva 43062,
Germany) purified vector-free 8-kb HPV inserts were labelled with [u-32P]dTTP
or [32P]dCTP (Amersham, England, or Bhabha Atomic Research Centre
(BARC), Bombay, India) using the standard procedures of nick translation
(Rigby et al., 1977) or random priming (Feinberg and Vogelstein, 1983). The
specific activity was greater than l-2 x lo8 cpm/ug DNA.
66

Southern blot hybridization (SBH)

Southern blotting procedures were essentially as described by Southern

(1975). Briefly, 67 ng of DNA (60-80 ul) was digested with 40 to 50 U of
BamHI or PstI (Boehringer Mannheim, Germany) overnight at 37°C
fractionated in 1% agarose gel and transferred onto Gene-screen plusTM
membrane (DuPont, NEN, U.S.A.). Hybridization was carried out under
stringent conditions (T,,, -20°C) with hybrid mix containing 50% formamide,
5 x SSC, 0.02% Denhardt’s solution, 0.25 ug/ml tRNA, 50 mM Na3P04 and
1% SDS. Hybridization was performed with two sets of ‘*P-1abelled HPV
probes, first with HPV 16 + 18 DNA and later with HPV 6 + 11 DNA.

Dot blot hybridization (DBH)

80 ul of DNA (6-7 ug from each specimen was denatured at 65°C for 30 min.
The extract was chilled immediately on ice and then blotted onto nitrocellulose
membrane using a dot blot apparatus Minifold I of Schleicher and Schuell
(Germany). After baking at 80°C for 1 h the membrane was hybridized under
stringent conditions using 32P-labelled HPV 6 + 11 and 16 + 18 DNA probes
separately.

Filter-in-situ hybridization (FISH)

The remaining 3 ml PBS solution containing scraped cervical cells were used
in FISH. The cells were filtered directly on to cellulose nitrate filter (Sartorius
type 11307, pore size 0.2 urn, Germany) using a pressure filtration unit of
Schleicher and Schuell. After denaturing and neutralizing for 5 min each, the
filters were dried and baked at 80°C for 1 h. Prehybridization was for 24 h at
42°C in prehybrid mix containing 50% formamide, 5 x SSC, 0.02 Denhardt
solution, 0.5 ug/ml tRNA and 50 mM trisodium phosphate Na3P04.
Hybridization was performed under stringent conditions (T, - 20°C) and
without SDS using 32P-labelled HPV probes. One half of each filter was
hybridized with HPV 6 + 11 DNA while the other half with HPV 16 + 18
probes. Following stringent washings at 68°C the Iilters were exposed to X-ray
film for 7 days.

Polymerase chain reaction (PCR)

The amplification of HPV DNA sequences was carried out using HPV-16-
specific primers using the method of Saiki et al. (1988) with some
modifications. The primers used were:
(Pl) 5’-AAGGCCAACTAAATGTCAC-3’
and (P2) 5’-CTGCTTTTATACTAACCGG-3’
These two oligo primers allow amplification of the conserved upstream
regulatory region (URR) of HPV 16 between nucleotides 7765 and 75. The
oligos were synthesized in an Applied Biosystems DNA synthesizer (model
381A, U.S.A.) and purified by NAP-10 columns (Pharmacia, U.S.A.).
Briefly, the method involves 50 ~1 of the reaction mix in a 0.5-ml Eppendorf
tube containing 10 mM Tris-HCl (pH 8.3) 1.5 mM MgC12, 50 mM KCl, and 75
ug/ml BSA with deoxyribonucleotides at final concentrations of 200 uM each
and primers at 1 uM and 2.5 U Taq DNA polymerase (Perkin Elmer Cetus,
U.S.A.), (Chien et al., 1976; Erlich et al., 1988). The reaction mixture was
overlayered with 50 ul of mineral oil (Cetus) and the amplifications were
carried out in a DNA Thermal Cycler (Perkin Elmer Cetus). About 0.5 ug of
cellular DNA was denatured at 94°C for 5 min, annealed at 55°C for 1 min
followed by extension for 2 min at 72°C. These steps were repeated for 35
additional cycles with denaturation at 94°C for 1 min only. In the last cycle the
extension at 72°C was carried out for 7 min. 15 to 20 ul of amplified product
was run in 3% Nusieve-agarose gel (2% Nusieve and 1% agarose) stained with
ethidium bromide and photographed under a UV transilluminator.

Results

Fig. 1 shows ethidium-bromide-stained agarose gel electrophoresis of 6

DNA samples (lanes l-6), each with 10 ul of extracted DNA, which amounts to
more than 0.8 pg. The first lane was a HindIII-digested 2 ug standard Lambda-
DNA marker (Boehringer, Germany). The quantity as well as quality of DNA
was found to be not inferior to that obtained by conventional methods. The

kb Xl 23456

Fig. I. Ethidium-bromide-stained agarose gel electrophoresis picture showing quantity as well as quality of
DNA extracted. Each lane contained IO pl of DNA solution.
68

a b
HPV
kb 16 I 2 3 4 5 6 7 8 9 JO kb

23.1-
9.5 - - 2.8
6.6 -
- 1.9
4.3 -
- 1.6
2.3 -
2.0 - - 1.0

1.3 -
- 0.5
Fig. 2. Southern blot analysis of DNA from cervical scrapes digested with (a) BarnHI and (b) PstI and
hybridized with 72P-labelled HPV 16 DNA. Lanes I, 2, 6, 7 and IO are showing a single 8-kb band. The
characteristic PstI fragments for HPV 16 are seen in (b).

yield of DNA is more than 95%, which is significantly higher than what is
normally recovered from the conventional phenol-chloroform method of
extraction. This was confirmed by simultaneous extraction of DNA from the

Fig. 3. Filter-in-situ hybridization (FISH) analysis of IO cervical scrape samples with Z2P-labelled HPV
l6+ 18 DNA probe. Sample numbers I and 6 are positive (serial Nos. 9 and IO in Table I).
 69

bp ox174 1 2 3 4 5

Fig. 4. Amplification of HPV 16 oligo primers in polymerase chain reaction (PCR) using 5 pl of extracted
DNA. 20 PI PCR products are showing amplification of214-bp product (arrow) after 3% Nusieve-agarose gel
electrophoresis, ethidium bromide staining and UV illumination. Lanes 3,4 and 5 are positive for HPV 16.

same amount of scraped cervical cells by both the methods and comparison of
DNA concentrations with a number of known DNA samples (data not shown).
Since further purification of DNA was not carried out to eliminate enzyme
digestion products, neither was spectrophotometric analysis. The DNAs
extracted by the present method were found to be sufficiently clean and pure
as they were readily digested by restriction enzymes. One to five units of
restriction enzymes per microgram of DNA was sufficient and no additional
amount of enzyme was required for complete digestion. Fig. 2a shows an 8-kb
band of HPV 16 in 5 of 10 DNA samples (lanes 1, 2, 6, 7, 10) after digestion
with a single-cut enzyme BarnHI. Bands in lanes 6 and 7 are very light and not
clearly visible because of the low copy number of the virus. The DNA of
sample 1 which was again cleaved with a multicut enzyme, PstI, and shows
characteristic PstI restriction fragments (arrows) for HPV 16 is presented in
Fig. 2b. The DNA showed good amplification of HPV 16 DNA sequences in
the polymerase chain reaction (see Fig. 4).
The results obtained by Southern blot, dot blot and filter in-situ
hybridization are shown in Table 1. Out of 10 samples, 5 (50%) were found
positive by Southern (see Fig. 2, a and b) and 4 (40%) by dot blot hybridization
(figures not shown) while only 2 (20%) were positive by FISH (Fig. 3). In the
PCR test, 7 (70%) cases were positive, which also included 5 Southern blot
positive samples. Fig. 4 shows PCR results from 5 specimens of which 3 were
clearly positive for HPV 16 DNA sequences. The expected size of the amplified
product is 214 bp which can easily be seen in Fig. 4.
TABLE I
Detection of human papillomavirus (HPV) DNA sequences in cervical scrapes by four different molecular biology techniques

Serial Case Cytodiagnosis Filter in situ Southern blot Dot blot PCR
No. No.
HPV

l6\18 6\ll 16\18 6\11 16\18 6\ll I6

I D-8 Control -ve -ve -ve -ve -ve -ve -ve

2 K-225 Control -ve -ve -ve -ve -ve -ve -ve
3 D-1008 Control -ve -ve -ve -ve -ve -ve fve
4 M-568 Mild. dysp. -ve -ve -ve -ve -ve -ve -ve
5 D-2834 Mild. dysp. -ve -ve +ve fve fve +ve fve
6 G-1082 Mild. dysp. -ve -ve -ve -ve -ve -ve + ve
: S-2939 Mod. dysp -ve -ve +ve -ve -ve -ve +ve
M-3710 Sev. dysp. -ve -ve +ve tve fve +ve +ve
9 K-2487 Inv. Ca. +ve fve +ve fve +ve +ve fve
IO M-382 I Inv. Ca. fve +ve +ve +ve fve fve + ve

Total 2110 2110 S/IO 4/lO 4/lO 4/lO 7110

(20%) (20%) (50%) (40%) (40%) (40%) (70%)
 71

Discussion

The quality as well as quantity of DNA, as estimated by agarose gel

electrophoresis following extraction by the present non-organic method, is
found to be not inferior to that obtained by conventional methods. The
concentration of DNA was determined by comparison with known amounts of
standard DNA samples in ethidium-bromide-stained gels. A UV spectro-
photometric analysis of DNA concentration was not done since the proteinase
K digestion products were also present in the DNA. The present method is very
rapid, easy and ideal for safe isolation of DNA from a large number of
specimens at a time. The entire procedure takes less than three hours and, as it
is carried out in a single Eppendorf tube, is economic. This also eliminates any
chance of error during handling of multiple specimens. Proteinase K works
efficiently on denatured protein at high temperature (60-65°C) and following
prolonged incubation at this temperature becomes autoinactivated (Jeanpierre,
1987). Because of this autolytic activity, the enzyme almost disappears from the
DNA solution (Jeanpierre, 1987) and the leftover enzyme-digestion products
do not interfere in further reactions.
Because of the high yield of DNA (> 95%) in less than one half of a single
scraped sample, it is possible to perform at least four different molecular tests
on the same specimen. This allows verification of the extent of sensitivity,
specificity and reliability of each test in diagnosing the HPV infection and, at
the same time it offers an opportunity for conducting other additional
investigations. In the present study, instead of conducting a single test of FISH,
three more molecular tests, namely dot blot, Southern blot, and the polymerase
chain reaction could also be carried out (see Figs. 2a,b, 3 and 4). The results
obtained (Table 1) indicate that FISH is less sensitive and reliable than other
methods. Out of 10 specimens tested only 2 (20%) were clearly positive by
FISH while 5 (50%) were positive by Southern and 4 (40%) by dot blot
hybridization. As expected the PCR test, which is at least a million times more
sensitive than Southern blotting and has potential for the detection of small
amounts of HPV DNA sequences present in clinical specimens, revealed 7
(70%) positive cases. The order of sensitivity of tests thus appears as FISH <
dot blot < Southern blot < PCR.
Whatever the test, the DNA isolated by the present method can be readily
used. This is routinely used in our laboratory for preparation of DNA for dot/
Southern blotting or PCR. More than a thousand specimen have been prepared
and as many as nine different restriction enzymes have been tested. The DNA is
also suitable for many other enzymatic analyses such as ligation, cloning,
sequencing and kinasing. The present method of DNA isolation can also be
efficiently employed for extraction of DNA from cultured cells, cell lines,
blood, amniotic/acytic fluid and bone marrow or fine needle aspirates.
72

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