Journal of Virological Methoris, 36 (1992) 63-12 0 1992 Elsevier Science Publishers B.V. / All rights reserved / Ol66-0934/92/%03.50
63
VIRMET 01272
A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection V. Gopalkrishna, Division
of Molecular
Adeline
Oncology,
Francis,
J.K. Sharma
Institute I$ Cytology New Delhi, India
(Accepted 4 September
and Preventive
and B.C. Das Oncology
(ICMR),
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; situ hybridization; Polymerase chain reaction
Southern blotting; Filter in
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 phenolchloroform 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-16specific 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 LambdaDNA 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 DNA extracted. Each lane contained
picture showing quantity IO pl of DNA solution.
as well as quality
of
68
b
a kb
HPV 16 I
2
3
4
5
6
7
8
9
JO
kb
23.19.5 -
- 2.8
6.6 4.3 2.3 2.0 1.3 -
- 1.9 - 1.6 - 1.0 - 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.
Total
9 IO
I 2 3 4 5 6 :
Serial No.
D-8 K-225 D-1008 M-568 D-2834 G-1082 S-2939 M-3710 K-2487 M-382 I
Case No.
Control Control Control Mild. dysp. Mild. dysp. Mild. dysp. Mod. dysp Sev. dysp. Inv. Ca. Inv. Ca.
Cytodiagnosis
Detection of human papillomavirus
TABLE I
-ve -ve -ve -ve
-ve -ve
-ve -ve -ve -ve fve -ve -ve fve +ve fve
4/lO (40%)
-ve -ve -ve -ve
fve -ve -ve tve fve +ve
4/lO (40%)
+ve
S/IO (50%)
+ve 2110 (20%)
2110 (20%)
fve
+ve +ve +ve
+ve
-ve
-ve
-ve
-ve +ve fve
-ve
-ve -ve
-ve
-ve
-ve
-ve
16\18
Dot blot
6\11
16\18
Southern blot
-ve -ve -ve
-ve
6\ll
l6\18
HPV
Filter in situ
4/lO (40%)
+ve fve
+ve
-ve
+ve -ve
-ve
-ve
-ve -ve
6\ll
(HPV) DNA sequences in cervical scrapes by four different molecular biology techniques
7110 (70%)
+ve fve + ve
+ve
-ve -ve fve -ve fve + ve
I6
PCR
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 spectrophotometric 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
References Das, B.C., Sehgal, A., Murthy. N.S., Gopalkrishna. V., Sharma, J.K., Das. D.K.. Singh, V. and Luthra, U.K. (1989) Human Papillomavirus and cervical cancer in Indian women. Lancet ii. 1271. de Villiers, E.M. (1989) Heterogeneity of the Human Papillomavirus group. J. Viral. 48984903. Chien. A., Edgar, D.P. and Trela, J.N. (1976) Deoxyribonucleic acid polymerase from the extreme thermostable Thermus aquaticus. J. Bacterial. 127. 1550&1557. Erlich, H.A., Gelfand, D.H. and Saiky, R.H. (1988) Specific DNA amplification. Nature (London) 331, 461462. Feinberg. A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anat. Biochem. 132, 6-l 1. Gissmann, L., Durst, M., Oltersdorf, T. and von Knebel, D.M. (1987) Human papillomaviruses and cervical cancer. Cancer cells 5, 275-280. Jeanpierre, M. (1987) A rapid method for the purification of DNA from blood. Nucleic Acids Res. 22, 961 I. Rigby, P.W.J., Diekmann. M., Rhodes, C. and Berg. P. (1977) Labelling deoxyribonucleic acid to high specific activity by nick-translation with DNA polymerase 1. J. Mol. Biol. 113, 237-242. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J.. Higuchi, R., Horn, G.T.. Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239. 487491. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98. 503-518. Wagner, D., Ikenberg. H.. Boehm, N. and Gissmann, L. (1984) Identification of human papillomavirus in cervical swabs by deoxyribonucleic acid in situ hybridization. Obstet. Gynecol. 64, 7677772. Wickenden, C., Steele, A., Malcolm, A.D.B. and Coleman. D.V. (1985) Screening for wart virus infection in normal and abnormal cervices by DNA hybridization of cervical scrapes. Lancet i, 65567. zur Hausen. H. (1989) Papillomaviruses as carcinomaviruses. Adv. Viral Oncol. 8, l-25.
63
VIRMET 01272
A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection V. Gopalkrishna, Division
of Molecular
Adeline
Oncology,
Francis,
J.K. Sharma
Institute I$ Cytology New Delhi, India
(Accepted 4 September
and Preventive
and B.C. Das Oncology
(ICMR),
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; situ hybridization; Polymerase chain reaction
Southern blotting; Filter in
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 phenolchloroform 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-16specific 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 LambdaDNA 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 DNA extracted. Each lane contained
picture showing quantity IO pl of DNA solution.
as well as quality
of
68
b
a kb
HPV 16 I
2
3
4
5
6
7
8
9
JO
kb
23.19.5 -
- 2.8
6.6 4.3 2.3 2.0 1.3 -
- 1.9 - 1.6 - 1.0 - 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.
Total
9 IO
I 2 3 4 5 6 :
Serial No.
D-8 K-225 D-1008 M-568 D-2834 G-1082 S-2939 M-3710 K-2487 M-382 I
Case No.
Control Control Control Mild. dysp. Mild. dysp. Mild. dysp. Mod. dysp Sev. dysp. Inv. Ca. Inv. Ca.
Cytodiagnosis
Detection of human papillomavirus
TABLE I
-ve -ve -ve -ve
-ve -ve
-ve -ve -ve -ve fve -ve -ve fve +ve fve
4/lO (40%)
-ve -ve -ve -ve
fve -ve -ve tve fve +ve
4/lO (40%)
+ve
S/IO (50%)
+ve 2110 (20%)
2110 (20%)
fve
+ve +ve +ve
+ve
-ve
-ve
-ve
-ve +ve fve
-ve
-ve -ve
-ve
-ve
-ve
-ve
16\18
Dot blot
6\11
16\18
Southern blot
-ve -ve -ve
-ve
6\ll
l6\18
HPV
Filter in situ
4/lO (40%)
+ve fve
+ve
-ve
+ve -ve
-ve
-ve
-ve -ve
6\ll
(HPV) DNA sequences in cervical scrapes by four different molecular biology techniques
7110 (70%)
+ve fve + ve
+ve
-ve -ve fve -ve fve + ve
I6
PCR
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 spectrophotometric 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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