Biol. Chem. Hoppe-Seyler Vol. 371, pp. 939-951, October 1990
Non-radioactive Labeling and Detection of Nucleic Acids III. Applications of the Digoxigenin System 3 Rudolf SEiBL3, Hans-Joachim , Rüdiger RÜGER 3 , Alfons MEiNDLb, Hans G. ZACHAI/ Rudolf RAßHOFER c , Michael ROGGENDORFC, HansWoLFc, Norbert ARNOLD**, Johannes WiENBERGd and Christoph KESSLER* a b c d
Boehringer Mannheim GmbH, Biochemisches Forschungszentrum Penzberg/Tutzing Institut für Physiologische Chemie, Universität München Max-von-Pettenkofer Institut, Universität München Institut für Anthropologie und Humangenetik, Universität München
(Received 5 April 1990)
Summary: The digoxigenin-based non-radioactive DNA labeling and detection system was applied in various hybridization protocols using digoxigeninlabeled probes obtained by enzymatic incorporation of Dig-[ll]-dUTP. In genomic blots single-copy genes (human tissue-type plasminogen activator, constant part of immunoglobulin light chain) can be detected with only 0.5 to 5 g human DNA depending on the type of probe and the length of the hybridizing reg-
ion. Due to its high sensitivity and specificity, the digoxigenin system is also appropriate for colony-, plaque-, and in situ hybridizations with metaphase chromosome spreads and fixed cells. Especially in the latter applications it is of great advantage, that with the digoxigenin system any significant background or unspecific side reactions with biological materials are avoided.
Nicht-radioaktive Markierung und Detektion von Nudeinsäuren: III. Anwendungen des Digoxigenin-Systems Zusammenfassung: Im Rahmen des nicht-radioaktiven DNA-Markierungs- und Detektionssystems auf Digoxigenin-Basis wurden Digoxigenin-markierte Sonden, die durch enzymatischen Einbau von Dig[ll]-dUTP erhalten wurden, in verschiedenen Hybridisierungsformaten eingesetzt. In genomischen Blots können einzelne Gene (Human-gewebsspezifischer Plasminogen-Aktivator, konstanter Teil der leichten -Kette von Immunglobulin) abhängig von der Art der Sonde und der Länge der hybridisierenden Re-
gion in nur 0.5 bis 5 % Human-DNA nachgewiesen werden. Wegen seiner hohen Sensitivität und Spezifität ist das Digoxigenin-System auch für Kolonie-, Plaque- und In-situ-Hybridisierungen an MetaphaseChromosomenpräparationen und fixierten Zellen geeignet. Besonders in den letztgenannten Anwendungen ist von großem Vorteil, daß mit dem DigoxigeninSystem ausgeprägter Hintergrund oder unspezifische Nebenreaktionen in biologischen Materialien vermieden werden.
Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1); Proteinase K, Tritirachium alkaline proteinase (EC 3.4.21.14); RNase, pancreatic ribonuclease (EC 3.1.27.5); Taq DNA polymerase, deoxynucleoside-triphosphaterDNA nucleotidyltransferase (DNA-directed) (from Thermus aquaticus) (EC 2.7.7.7). Abbreviations: BCIP, 5-bromo-4-chloro-3-indoIyl phosphate; CHO, Chinese hamster ovary cells; CIAP, calf intestine alkaline phosphatase; DEPC, diethyl pyrocarbonate; Dig, digoxigenin; (Dig), digoxigenin-specific sheep polyclonal antibody (Fab-fragments); EBV, Epstein Barr virus; EDTA, ethylenediamine tetraacetic acid, disodium salt; HBV, Hepatitis B virus; MOPS, 3-[N-morpholino]propanesulfonic acid; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; S A, streptavidin; SDS, sodium dodecyl sulphate (lauryl sulfate, sodium salt); Taq, Thermus aquaticus; SSC, standard saline citrate (0.15\i NaCl, 0.015M Na citrate, pH 7.0); SET, Tris/NaCl/EDTA buffer, see Material and methods, paragraph 3;Tris, tris[hydroxymethyl]aminomethane; t-PA, tissue-type plasminogen activator.
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940
R. Seibietal.
Vol. 371 (1990))
Key words: Digoxigenin system, blot formats, colony hybridization, plaque hybridization, in situ hybridization, metaphase chromosomes.
Enzymatic labeling of DNA by nick translation with biotin-labeled nucleotides and subsequent detection of the biotin-labeled DNA with streptavidin-alkaline phosphatase conjugate has been the best known and the most sensitive method of non-radioactive DNA labeling and detection for several years^~6l In our efforts to improve the applicability of non-radioactive systems we found that replacement of biotin by digoxigenin and of streptavidin by a digoxigenin-specific antibody resulted in similar sensitivities but in a remarkable reduction of unspecific background reactions171. With respect to published protocols11'6^ we were able to increase the sensitivity in both the biotin and the digoxigenin system by exchanging nick translation to random-primed labeling and by using optimized conjugates181. In addition, in both systems unspecific background reactions could further be suppressed by the addition of an improved blocking reagent to the pre- and probe-hybridization solution as well as to the solution used for pretreatment of the membrane prior to the detection reaction in order to prevent unspecific binding of the conjugate. The optimized protocol for the digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot-and Southern-blots without any significant background^101. In this article we show several applications in more complex hybridization approaches: detection of single-copy genes in human genomic DNA, colony hybridization, plaque hybridization, detection of hepatitis B virus DNA in human sera of infected patients by slot-blot analysis, detection of Epstein-Barr virus sequences in DNA of latently infected cells, and in situ hybridizations with fixed cells and on chromosome spreads. We further show the application of labeling of nucleic acids with digoxigenin during polymerase chain reaction.
Materials and Methods A) Detection of single-copy genes in genomic DNA 1) Preparation of DNA blots High molecular mass DNA was prepared according to Blin and Stafford'11'. The purified DNA was digested with the appropriate restriction enzyme (Boehringer Mannheim) according to the instructions of the manufacturer, run on 1% (w/v) agarose gels, blotted with 20 x SSC overnight either onto nitrocellulose filters BA85 (Schleicher & Schuell) or nylon membranes Hybond N (Amer-
sham). For DNA fixation the nitrocellulose filters were incubated for 2 h at 80 °C in a vacuum oven; in case of nylon membranes the DNA was UV-crosslinked to the membranes. 2) Labeling of the DNA probes The appropriate DNA fragment was eluted from agarose gels after restriction enzyme digestion and electrophoresis. Labeling of 0.51 μ-g of denatured DNA fragments with digoxigenin was performed with the DNA labeling and detection kit, non-radioactive (Boehringer Mannheim) as described'71. Biotin-labeling was performed with biotin-[16]-dUTP (Boehringer Mannheim) and the appropriate components of the randomprimed DNA labeling kit (Boehringer Mannheim). With biotin[16]-dUTP the sensitivity was increased as compared to biotin-[ll]dUTP. The reaction conditions were handled as for the incorporation of digoxigenin but with a different concentration of biotin-[16]dUTP in relation to dTTP and an increase in reaction time. For a typical labeling reaction to 1 μg DNA were added: 2 μΐ dATP, 0.5mM; 2 μΐ dCTP, O.SmM; 2 μΐ dGTP, 0.5mM; 1.6 μΐ dTTP, 0.5mM; 1.6 μΐ biotin-[16]-dUTP, 0.3mM; 2 μΐ 10 x concentrated randomprimed reaction mixture and 1 μΐ Klenow enzyme (2 υ/μ./) in a total volume of 20 μΐ. The reaction was incubated for at least 1.5 h at 37 °C. Digoxigenin- and biotin-labeled DNAs were purified by ethanol precipitation as described'71. Radioactive labeling of the DNA fragments was performed with the random-primed DNA labeling kit (Boehringer Mannheim) and [a-32P]dCTP (Amersham.> 3000 Ci/mmol). 50-100 ng of the DNAfragments were incubated with 1 μΐ each of dATP, dGTP and dTTP, 2 μΐ reaction mixture, 100 μα [a-32P]dCTP and 1 μΐ Klenow enzyme for l h at 37 °C. Labeled DNA was separated from unincorporated radioactivity by chromatography with Sephadex G50 (Pharmacia). 3) Hybridization Non-radioactive hybridization with biotin- and digoxigenin-labeled probes was according to the protocol for the DNA labeling and detection kit, non-radioactive (Boehringer Mannheim) and as described'71. The filters were pre-hybridized (superior for both digoxigenin- and biotin-labeled probes) for 1-2 h at 68 °C with a hybridization solution of 5 x SSC (Na-citrate, 75mM; NaCl, 750mM; pH7.0/25°C); AMauroyl-sarcosin, 0.1% (w/v); SDS, 0.02% (w/v); blocking reagent, 0.5% (w/v). Probe-hybridization took place for 16 h at 68 °C in hybridization solution containing either 26 ng/m/ digoxigenin-labeled DNA or 13 ng/m/biotin-labeled DNA (for determination of the amount of labeled DNA see Table 1 of ref.'81). Usually 100 ng and 50 ng of unlabeled template DNA were also present with the 26 ng digoxigenin- and 13 ng biotin-labeled probes respectively. The filters were washed 2 x 5 min at room temperature with 2 x SSC, 0.1% (w/v) SDS and 2 x 15 min at 68 °C with 0.1 x SSC; SDS, 0.1% (w/v). For the radioactive experiments the filters were pre-hybridized 2 h at 68 °C in 4 x SET(Tris/HCl, 120mM; NaCl, 0.6M; EDTA, 4mM; pH 7.0/25 °C) or 4 x SSC; AMauroylsarcosine, 1% (w/v); SDS, 0.2% (w/v); 4 x Denhardt's solution [ficoll, 1% (w/v); polyvinylpyrrolidone, 1% (w/v); bovine serum albumin, 1% (w/v)]; 1 x Denhardt's solution was used in the experiments shown in Fig. 5. Hybridization was performed for 16 h at 68 °C in 4 x SET or 4 x SSC; AMauroylsarcosine, 1% (w/v); SDS, 0.2% (w/v); 4 x Denhardt's solution with 2-5 x 108 cpm^g DNA. All filters were washed at room temperature with 2 x SSC; SDS, 0.1% (w/v), and at 68 °C with 0.1 x SSC; SDS, 0.1% (w/v).
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Vol.371 (1990)
Applications of the Digoxigenin System
4) Detection Detection of the digoxigenin- and biotin-labeled DNA was according to the protocol of the DNA labeling and detection kit, nonradioactive (Boehringer Mannheim) and as described in ref.[7). After washing and blocking with blocking reagent, 0.5% (w/v), the filters were subsequently incubated for 30 min with anti-digoxigenin-antibody (Fab): alkaline phosphatase conjugate ((Dig) :CIAP) solution freshly diluted to 150 mU/m/as described in ref.'7' for the digoxigenin-labeled DNA, and streptavidin:alkaline phosphatase conjugate (SA:CIAP) solution freshly diluted to 150 mU/m/ as described in ref.'7' for the biotin-labeled DNA. Colour reaction was allowed to run routinely for 16 h; especially with higher amounts of blotted DNA the colour precipitate was already visible after about 1 h.The radioactively labeled filters were dried and exposed over-night at - 70 °C with two intensifying screens (DuPont) to a X-OMAT S X-ray film (Kodak).
B) Colony hybridization Bacteria were transferred to or directly plated onto nitrocellulose filters lying on agarose plates according to^12'. After incubation for 8 h (5-10 h) at 37 °C the nitrocellulose filters were pulled off and placed for 2 x 5 min on filter papers saturated with NaOH, 0.5M. For the following neutralization reaction the filters were placed for 2 x 5 min on filter papers saturated withTris/HCl, IM; pH 7.4/25 °C and 1 x 5 min on paper withTris/HCl, 0.5M; NaCl, I.SM; pH 7.4/ 25 °C. The filters were pressed between two dry filter papers, air dried and baked for 2 h at 80 °C in a vacuum oven. Labeling of the DNA, pre- and probe-hybridization of the filters and immunological detection of the hybrids was performed as described in Materials and Methods, section A.
C) Plaque hybridization λ plaques were transferred to nitrocellulose filters as described by Benton and Davis^13'. Dry nitrocellulose filters were placed on the agar plates with the plaques. After adsorption the filters were carefully lifted followed by alkali treatment and subsequent immobilization, hybridization and detection steps, as described in Materials and Methods, section B.
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1.2) Labeling, hybridization and detection A 1.8 kb amHI-fragment from cloned hepatitis B virus subtype adw (position 1392 to 30)[14' was eluted from agarose gels. Nonradioactive labeling of the amHI-fragment by random-primed incorporation of Dig-[ll]-dUTP, hybridization with the digoxigeninlabeled probe and final detection of the digoxigenin-label within the hybrid was performed as described in Materials and Methods, section A.2-4. In case of radioactive hybridization using [a-32P]dCTP for random-primed labeling of the oraHI-fragment, the hybridization conditions described by Scotto et al.'15' were modified as follows: The membrane was pre-hybridized at 68 °C for 3 h in 5 x Denhardt's solution; SDS, 0.5% (w/v); 6 x SSC; tRNA, 400 μg/m/ (Boehringer Mannheim). Probe-hybridization was performed at the same temperature in the same solution (50 μΐΐcm2 filter area) for at least 16 h with 5 χ 106 cpm of the radioactive probe per ml hybridization mixture. After hybridization the membranes were washed for 15 min at room temperature in 2 x SSC; SDS, 0.5% (w/v), and twice for 45 min at 68°C in 0.1 x SSC; SDS, 0.5% (w/v). The filters were air-dried, covered with plastic foil and exposed for at least 16 h at - 70 °C with two intensifying screens (DuPont) and a X-OMAT S X-ray film (Kodak).
2) Detection of Epstein Barr virus DNA by Southern-blot Two well characterized cell lines were used as hybridization samples. BJ AB cells were derived from a human B-cell lymphoma free of EBV genomes. Raji cells were derived from a human Burkitt's lymphoma and contain about 50 EBV genomes per cell.The preparation of the DNA blots was performed as described in Materials and Methods, section A.I; the non-radioactive labeling with digoxigenin and biotin as well as the radioactive labeling with 32P as described in Materials and Methods, section A.2. Non-radioactive hybridization with either biotin- or digoxigenin-labeled probes and subsequent detection was performed as described in Materials and Methods, section A.3,4. 50 ng of biotin- and digoxigenin-labeled DNA/m/ hybridization solution were used as probes. The specific activity of the 32P-labeled probe was 2.5 x 108 cpm//z.g. Hybridization with the 32P-labeled probe was performed essentially as in Materials and Methods, section D.I, but with 50% (v/v) formamide in hybridization solution at 50 °C.
3) Detection of Epstein-Barr viral DNA by in situ hybridization D) Detection of viral DNA 1) Detection of hepatitis B virus DNA in human sera 1.1) Preparation of the filters Sera of hepatitis B virus-infected and noninfected persons were kindly provided by the diagnostic department of the Max-von-Pettenkofer Institute, D-8000 Munich. Serological tests for HBV markers (HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe and anti-HBc!gM) were performed with radioimmunoassays using commercially available reagent kits (Abbott Laboratories). Biodyne A nylon membranes, pore size 1.2 μ-m (Pall) were prepared by floating for 5 min in H2O dest., and for 15 min in 10 x SSC. After air drying, the membranes were placed onto Whatman 3MM paper soaked with 20 x SSC and adjusted in a minifold apparatus (Schleicher & Schuell). 100 μΐ of each serum sample was centrifuged in a benchtop centrifuge for 5 min, incubated with 300 μΐ NaOH, 0.5M for 5 min at room temperature and again centrifuged for 5 min. Finally 200 μΐ of the alkali-denatured serum was loaded per well of the minifold slot-blot apparatus and the solution was passed through the membrane applying a water-suction pump. The membranes were removed from the slot-blot apparatus and neutralized by floating 5 min on a solution of Na-acetate, 3M; pH 5.0/25 °C and 2 x 5 min on a solution of 2 x SSC.The filters were air dried and baked 2 h at 80 °C in a vacuum oven.
3.1) Fixing of suspension cells Suspension cells (B95-8 and BJAB) were washed in PBS (phosphate buffered saline: Na/K-phosphate, lOmM; KC1,2.7mM; NaCl, 120mM; pH 7.2/25 °C) after the fourth day of splitting and dropped on precoated slides. B95-8 cells were derived from a marmoset B-lymphoid cell line immortalized by EBV. Each individual B95-8 cell contains a few EBV genomes per cell with restricted expression of viral genes. 5-10% of the cells spontaneously enter a lytic cycle of viral replication; these cells contain up to 10000 viral genomes per cell. Degreased slides were baked for l h at 100 °C, incubated for 3 h at 65 °C in 3 x SSC; ficoll, 0.02% (w/v); polyvinylpyrrolidone, 0.02% (w/v); bovine serum albumin, 0.02% (w/v), dipped shortly in H2O dest. and fixed in 3:1 (v/v) ethanol/acetic acid for 20 min at 22 °C.The size of the cell spot fitted under an 18 x 18 mm coverslip.The airdried cells on these slides were fixed for 2.5 min at 22 °C in freshly prepared paraformaldehyde, 4% (w/v) in PBS. 3.2) Preparation of digoxigenin-labeled probe 26 different EBV[£amHI]-fragments (not the EBV[BamHl]Kfragment due to cross-reaction with cellular DNA) were eluted from agarose gel after restriction enzyme digestion and electrophoresis, pooled and labeled with digoxigenin as described in Materials and Methods, section A.2.
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942
R. Seibiet al.
3.3) In situ hybridization Protocols were derived from Wolf et al.'16', Falser et al.' 17 ' and Heiles et al.'10' with some modifications. HC1 treatment as suggested by some authors was omitted, as it seemed to introduce false positive signals in some cases. The slides were washed twice for 10 min at 22 °C in PBS; MgCI2, 5mM, and incubated at 50 °C for 30 min in 2 χ SSC; EDTA, 5mM. After a 15 min treatment with 1 μ-g proteinase K/m/ PBS at 37 °C, the slides were washed for 10 min at 22 °C in PBS containing glycine, 0.02% (w/v), followed by post-fixation for 20 min at 22 °C with freshly prepared paraformaldehyde, 4% (w/v). Final washing was twice for 15 min at 22 °C with PBS; MgCl 2 ,5mM. Pre-hybridization was carried out for 15 min at 22 °C in 50 μ//5ροΙ of 6 x SSC; formamide, 45% (v/v); 5 x Denhardt's solution; heat-denatured salmon sperm DNA, 100 pg/ml. After careful removal of the pre-hybridization solution without washing 20 μ/ of probe-hybridization solution were placed under a siliconized coverslip containing 7.5 ng digoxigenin-labeled probe in 6 x SSC; formamide, 45% (v/v); 5 x Denhardt's solution; dextran sulfate, 10% (w/v). The edges were sealed with rubber cement (Fixogum, Marabu). The cellular and probe DNAs were denatured while placing the slides directly on a hot plate (Ortho Diagnostics) at 90-94 °C for 5 min. The slides were cooled on ice and incubated at 42 °C for 16 h. After the coverslips had been removed the slides were washed twice at 42 °C for 15 min in 6 x SSC; formamide, 45% (v/v), then twice at 22 °C for 5 min in 2 x SSC, and finally twice at 50 °C for 15 min in 0.2 x SSC. 3.4) Detection The detection reaction was carried out as described in Materials and Methods, section A.4.The colour reaction was stopped after 2-6 h withTris/HCl, 10mM; EDTA, ImM; pH 8.0/25 °C. No further staining was performed on the slides, they were evaluated with a differential interference contrast device (Zeiss).
E) In situ hybridization ofmetaphase chromosomes Cells were exposed to colcemid, 0.05 yug/m/, for l h before fixation. Chromosome preparation was performed as described in ref.'18'. In situ hybridization was performed according to the protocol described by Ambros et al.1191. To minimize non-specific binding of the probe, slides were treated with 100 μΐ of DNase-free RNase (100 /Ag/m/, Boehringer Mannheim) and sealed with a coverslip. After 1 h incubation at 37 °C, coverslips were removed and the slides were washed with water and dehydrated with alcohol. 50 μ,/ hybridization solution was placed on the slides under a coverslip, sealed with rubber cement, then chromosomes and the probe were denatured at 72-74 °C for 10 min. The hybridization solution was composed of 2 x SSC; formamide, 50% (v/v); dextran sulphate, 10% (w/v); sonicated salmon sperm DNA, 0.5 mg/m/; digoxigeninlabeled probe, 16 ng/m/. Hybridization was performed at 39 °C for 14 h. Nonspecific binding of labeled probe was avoided by appropriate hybridization conditions and by additional washing steps at a temperature adequate to remove adventiously bound probe. After removing the coverslips the slides were either washed at room temperature for 5 min with 2 x SSC or at 39-41 °C for 10-30 min with 2 x SSC; formamide, 50% (v/v), or alternatively three times at 37 °C for 5 min with 2 x SSC, one for 3 min with PBS;Triton X-100, 0.1% (v/v), and again three times for 5 min with PBS. For detection of hybridized digoxigenin-labeled probe the slides were washed withTris/HCl, lOOmM; NaCl, 150mM; pH 7.5/25 °C and incubated for 1 h at room temperature with diluted (Dig):CIAP-solution (150 mU/m/; Boehringer Mannheim). The slides were subsequently washed with Tris/HCl, lOOmM; NaCl, ISOmM; pH 7.5/25 °C, and then with Tris/HCl, lOOmM; NaCl, lOOmM; MgCl2, 50mM; pH 9.5/25 °C. Colour development was stopped after 1 h by washing the slides extensively in Tris/HCl, lOmM; EDTA, ImM; pH 8.0/25 °C. For microscopic analysis a Leitz
Vol. 371 (1990))
photomicroscope equipped with reflection-constrast'20' was used. Photographs were taken on Kodak Ektachrome andTechnical Pain film. F) Labeling of nucleic acids during polymerase chain reaction Serial dilutions of 10 ng to 0.1 pg from linearized plasmid pSPTlSrteo containing the neomycin gene (940 bp) were used a:s templates in polymerase chain reactions according to Saiki et al.'21j'. These plasmid concentrations correspond to 2.3 ng to 23 fg o>f neomycin-specific insert sequences. The PCR was primed with SP
Non-radioactive Labeling and Detection of Nucleic Acids III. Applications of the Digoxigenin System 3 Rudolf SEiBL3, Hans-Joachim , Rüdiger RÜGER 3 , Alfons MEiNDLb, Hans G. ZACHAI/ Rudolf RAßHOFER c , Michael ROGGENDORFC, HansWoLFc, Norbert ARNOLD**, Johannes WiENBERGd and Christoph KESSLER* a b c d
Boehringer Mannheim GmbH, Biochemisches Forschungszentrum Penzberg/Tutzing Institut für Physiologische Chemie, Universität München Max-von-Pettenkofer Institut, Universität München Institut für Anthropologie und Humangenetik, Universität München
(Received 5 April 1990)
Summary: The digoxigenin-based non-radioactive DNA labeling and detection system was applied in various hybridization protocols using digoxigeninlabeled probes obtained by enzymatic incorporation of Dig-[ll]-dUTP. In genomic blots single-copy genes (human tissue-type plasminogen activator, constant part of immunoglobulin light chain) can be detected with only 0.5 to 5 g human DNA depending on the type of probe and the length of the hybridizing reg-
ion. Due to its high sensitivity and specificity, the digoxigenin system is also appropriate for colony-, plaque-, and in situ hybridizations with metaphase chromosome spreads and fixed cells. Especially in the latter applications it is of great advantage, that with the digoxigenin system any significant background or unspecific side reactions with biological materials are avoided.
Nicht-radioaktive Markierung und Detektion von Nudeinsäuren: III. Anwendungen des Digoxigenin-Systems Zusammenfassung: Im Rahmen des nicht-radioaktiven DNA-Markierungs- und Detektionssystems auf Digoxigenin-Basis wurden Digoxigenin-markierte Sonden, die durch enzymatischen Einbau von Dig[ll]-dUTP erhalten wurden, in verschiedenen Hybridisierungsformaten eingesetzt. In genomischen Blots können einzelne Gene (Human-gewebsspezifischer Plasminogen-Aktivator, konstanter Teil der leichten -Kette von Immunglobulin) abhängig von der Art der Sonde und der Länge der hybridisierenden Re-
gion in nur 0.5 bis 5 % Human-DNA nachgewiesen werden. Wegen seiner hohen Sensitivität und Spezifität ist das Digoxigenin-System auch für Kolonie-, Plaque- und In-situ-Hybridisierungen an MetaphaseChromosomenpräparationen und fixierten Zellen geeignet. Besonders in den letztgenannten Anwendungen ist von großem Vorteil, daß mit dem DigoxigeninSystem ausgeprägter Hintergrund oder unspezifische Nebenreaktionen in biologischen Materialien vermieden werden.
Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1); Proteinase K, Tritirachium alkaline proteinase (EC 3.4.21.14); RNase, pancreatic ribonuclease (EC 3.1.27.5); Taq DNA polymerase, deoxynucleoside-triphosphaterDNA nucleotidyltransferase (DNA-directed) (from Thermus aquaticus) (EC 2.7.7.7). Abbreviations: BCIP, 5-bromo-4-chloro-3-indoIyl phosphate; CHO, Chinese hamster ovary cells; CIAP, calf intestine alkaline phosphatase; DEPC, diethyl pyrocarbonate; Dig, digoxigenin; (Dig), digoxigenin-specific sheep polyclonal antibody (Fab-fragments); EBV, Epstein Barr virus; EDTA, ethylenediamine tetraacetic acid, disodium salt; HBV, Hepatitis B virus; MOPS, 3-[N-morpholino]propanesulfonic acid; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; S A, streptavidin; SDS, sodium dodecyl sulphate (lauryl sulfate, sodium salt); Taq, Thermus aquaticus; SSC, standard saline citrate (0.15\i NaCl, 0.015M Na citrate, pH 7.0); SET, Tris/NaCl/EDTA buffer, see Material and methods, paragraph 3;Tris, tris[hydroxymethyl]aminomethane; t-PA, tissue-type plasminogen activator.
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940
R. Seibietal.
Vol. 371 (1990))
Key words: Digoxigenin system, blot formats, colony hybridization, plaque hybridization, in situ hybridization, metaphase chromosomes.
Enzymatic labeling of DNA by nick translation with biotin-labeled nucleotides and subsequent detection of the biotin-labeled DNA with streptavidin-alkaline phosphatase conjugate has been the best known and the most sensitive method of non-radioactive DNA labeling and detection for several years^~6l In our efforts to improve the applicability of non-radioactive systems we found that replacement of biotin by digoxigenin and of streptavidin by a digoxigenin-specific antibody resulted in similar sensitivities but in a remarkable reduction of unspecific background reactions171. With respect to published protocols11'6^ we were able to increase the sensitivity in both the biotin and the digoxigenin system by exchanging nick translation to random-primed labeling and by using optimized conjugates181. In addition, in both systems unspecific background reactions could further be suppressed by the addition of an improved blocking reagent to the pre- and probe-hybridization solution as well as to the solution used for pretreatment of the membrane prior to the detection reaction in order to prevent unspecific binding of the conjugate. The optimized protocol for the digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot-and Southern-blots without any significant background^101. In this article we show several applications in more complex hybridization approaches: detection of single-copy genes in human genomic DNA, colony hybridization, plaque hybridization, detection of hepatitis B virus DNA in human sera of infected patients by slot-blot analysis, detection of Epstein-Barr virus sequences in DNA of latently infected cells, and in situ hybridizations with fixed cells and on chromosome spreads. We further show the application of labeling of nucleic acids with digoxigenin during polymerase chain reaction.
Materials and Methods A) Detection of single-copy genes in genomic DNA 1) Preparation of DNA blots High molecular mass DNA was prepared according to Blin and Stafford'11'. The purified DNA was digested with the appropriate restriction enzyme (Boehringer Mannheim) according to the instructions of the manufacturer, run on 1% (w/v) agarose gels, blotted with 20 x SSC overnight either onto nitrocellulose filters BA85 (Schleicher & Schuell) or nylon membranes Hybond N (Amer-
sham). For DNA fixation the nitrocellulose filters were incubated for 2 h at 80 °C in a vacuum oven; in case of nylon membranes the DNA was UV-crosslinked to the membranes. 2) Labeling of the DNA probes The appropriate DNA fragment was eluted from agarose gels after restriction enzyme digestion and electrophoresis. Labeling of 0.51 μ-g of denatured DNA fragments with digoxigenin was performed with the DNA labeling and detection kit, non-radioactive (Boehringer Mannheim) as described'71. Biotin-labeling was performed with biotin-[16]-dUTP (Boehringer Mannheim) and the appropriate components of the randomprimed DNA labeling kit (Boehringer Mannheim). With biotin[16]-dUTP the sensitivity was increased as compared to biotin-[ll]dUTP. The reaction conditions were handled as for the incorporation of digoxigenin but with a different concentration of biotin-[16]dUTP in relation to dTTP and an increase in reaction time. For a typical labeling reaction to 1 μg DNA were added: 2 μΐ dATP, 0.5mM; 2 μΐ dCTP, O.SmM; 2 μΐ dGTP, 0.5mM; 1.6 μΐ dTTP, 0.5mM; 1.6 μΐ biotin-[16]-dUTP, 0.3mM; 2 μΐ 10 x concentrated randomprimed reaction mixture and 1 μΐ Klenow enzyme (2 υ/μ./) in a total volume of 20 μΐ. The reaction was incubated for at least 1.5 h at 37 °C. Digoxigenin- and biotin-labeled DNAs were purified by ethanol precipitation as described'71. Radioactive labeling of the DNA fragments was performed with the random-primed DNA labeling kit (Boehringer Mannheim) and [a-32P]dCTP (Amersham.> 3000 Ci/mmol). 50-100 ng of the DNAfragments were incubated with 1 μΐ each of dATP, dGTP and dTTP, 2 μΐ reaction mixture, 100 μα [a-32P]dCTP and 1 μΐ Klenow enzyme for l h at 37 °C. Labeled DNA was separated from unincorporated radioactivity by chromatography with Sephadex G50 (Pharmacia). 3) Hybridization Non-radioactive hybridization with biotin- and digoxigenin-labeled probes was according to the protocol for the DNA labeling and detection kit, non-radioactive (Boehringer Mannheim) and as described'71. The filters were pre-hybridized (superior for both digoxigenin- and biotin-labeled probes) for 1-2 h at 68 °C with a hybridization solution of 5 x SSC (Na-citrate, 75mM; NaCl, 750mM; pH7.0/25°C); AMauroyl-sarcosin, 0.1% (w/v); SDS, 0.02% (w/v); blocking reagent, 0.5% (w/v). Probe-hybridization took place for 16 h at 68 °C in hybridization solution containing either 26 ng/m/ digoxigenin-labeled DNA or 13 ng/m/biotin-labeled DNA (for determination of the amount of labeled DNA see Table 1 of ref.'81). Usually 100 ng and 50 ng of unlabeled template DNA were also present with the 26 ng digoxigenin- and 13 ng biotin-labeled probes respectively. The filters were washed 2 x 5 min at room temperature with 2 x SSC, 0.1% (w/v) SDS and 2 x 15 min at 68 °C with 0.1 x SSC; SDS, 0.1% (w/v). For the radioactive experiments the filters were pre-hybridized 2 h at 68 °C in 4 x SET(Tris/HCl, 120mM; NaCl, 0.6M; EDTA, 4mM; pH 7.0/25 °C) or 4 x SSC; AMauroylsarcosine, 1% (w/v); SDS, 0.2% (w/v); 4 x Denhardt's solution [ficoll, 1% (w/v); polyvinylpyrrolidone, 1% (w/v); bovine serum albumin, 1% (w/v)]; 1 x Denhardt's solution was used in the experiments shown in Fig. 5. Hybridization was performed for 16 h at 68 °C in 4 x SET or 4 x SSC; AMauroylsarcosine, 1% (w/v); SDS, 0.2% (w/v); 4 x Denhardt's solution with 2-5 x 108 cpm^g DNA. All filters were washed at room temperature with 2 x SSC; SDS, 0.1% (w/v), and at 68 °C with 0.1 x SSC; SDS, 0.1% (w/v).
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Vol.371 (1990)
Applications of the Digoxigenin System
4) Detection Detection of the digoxigenin- and biotin-labeled DNA was according to the protocol of the DNA labeling and detection kit, nonradioactive (Boehringer Mannheim) and as described in ref.[7). After washing and blocking with blocking reagent, 0.5% (w/v), the filters were subsequently incubated for 30 min with anti-digoxigenin-antibody (Fab): alkaline phosphatase conjugate ((Dig) :CIAP) solution freshly diluted to 150 mU/m/as described in ref.'7' for the digoxigenin-labeled DNA, and streptavidin:alkaline phosphatase conjugate (SA:CIAP) solution freshly diluted to 150 mU/m/ as described in ref.'7' for the biotin-labeled DNA. Colour reaction was allowed to run routinely for 16 h; especially with higher amounts of blotted DNA the colour precipitate was already visible after about 1 h.The radioactively labeled filters were dried and exposed over-night at - 70 °C with two intensifying screens (DuPont) to a X-OMAT S X-ray film (Kodak).
B) Colony hybridization Bacteria were transferred to or directly plated onto nitrocellulose filters lying on agarose plates according to^12'. After incubation for 8 h (5-10 h) at 37 °C the nitrocellulose filters were pulled off and placed for 2 x 5 min on filter papers saturated with NaOH, 0.5M. For the following neutralization reaction the filters were placed for 2 x 5 min on filter papers saturated withTris/HCl, IM; pH 7.4/25 °C and 1 x 5 min on paper withTris/HCl, 0.5M; NaCl, I.SM; pH 7.4/ 25 °C. The filters were pressed between two dry filter papers, air dried and baked for 2 h at 80 °C in a vacuum oven. Labeling of the DNA, pre- and probe-hybridization of the filters and immunological detection of the hybrids was performed as described in Materials and Methods, section A.
C) Plaque hybridization λ plaques were transferred to nitrocellulose filters as described by Benton and Davis^13'. Dry nitrocellulose filters were placed on the agar plates with the plaques. After adsorption the filters were carefully lifted followed by alkali treatment and subsequent immobilization, hybridization and detection steps, as described in Materials and Methods, section B.
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1.2) Labeling, hybridization and detection A 1.8 kb amHI-fragment from cloned hepatitis B virus subtype adw (position 1392 to 30)[14' was eluted from agarose gels. Nonradioactive labeling of the amHI-fragment by random-primed incorporation of Dig-[ll]-dUTP, hybridization with the digoxigeninlabeled probe and final detection of the digoxigenin-label within the hybrid was performed as described in Materials and Methods, section A.2-4. In case of radioactive hybridization using [a-32P]dCTP for random-primed labeling of the oraHI-fragment, the hybridization conditions described by Scotto et al.'15' were modified as follows: The membrane was pre-hybridized at 68 °C for 3 h in 5 x Denhardt's solution; SDS, 0.5% (w/v); 6 x SSC; tRNA, 400 μg/m/ (Boehringer Mannheim). Probe-hybridization was performed at the same temperature in the same solution (50 μΐΐcm2 filter area) for at least 16 h with 5 χ 106 cpm of the radioactive probe per ml hybridization mixture. After hybridization the membranes were washed for 15 min at room temperature in 2 x SSC; SDS, 0.5% (w/v), and twice for 45 min at 68°C in 0.1 x SSC; SDS, 0.5% (w/v). The filters were air-dried, covered with plastic foil and exposed for at least 16 h at - 70 °C with two intensifying screens (DuPont) and a X-OMAT S X-ray film (Kodak).
2) Detection of Epstein Barr virus DNA by Southern-blot Two well characterized cell lines were used as hybridization samples. BJ AB cells were derived from a human B-cell lymphoma free of EBV genomes. Raji cells were derived from a human Burkitt's lymphoma and contain about 50 EBV genomes per cell.The preparation of the DNA blots was performed as described in Materials and Methods, section A.I; the non-radioactive labeling with digoxigenin and biotin as well as the radioactive labeling with 32P as described in Materials and Methods, section A.2. Non-radioactive hybridization with either biotin- or digoxigenin-labeled probes and subsequent detection was performed as described in Materials and Methods, section A.3,4. 50 ng of biotin- and digoxigenin-labeled DNA/m/ hybridization solution were used as probes. The specific activity of the 32P-labeled probe was 2.5 x 108 cpm//z.g. Hybridization with the 32P-labeled probe was performed essentially as in Materials and Methods, section D.I, but with 50% (v/v) formamide in hybridization solution at 50 °C.
3) Detection of Epstein-Barr viral DNA by in situ hybridization D) Detection of viral DNA 1) Detection of hepatitis B virus DNA in human sera 1.1) Preparation of the filters Sera of hepatitis B virus-infected and noninfected persons were kindly provided by the diagnostic department of the Max-von-Pettenkofer Institute, D-8000 Munich. Serological tests for HBV markers (HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe and anti-HBc!gM) were performed with radioimmunoassays using commercially available reagent kits (Abbott Laboratories). Biodyne A nylon membranes, pore size 1.2 μ-m (Pall) were prepared by floating for 5 min in H2O dest., and for 15 min in 10 x SSC. After air drying, the membranes were placed onto Whatman 3MM paper soaked with 20 x SSC and adjusted in a minifold apparatus (Schleicher & Schuell). 100 μΐ of each serum sample was centrifuged in a benchtop centrifuge for 5 min, incubated with 300 μΐ NaOH, 0.5M for 5 min at room temperature and again centrifuged for 5 min. Finally 200 μΐ of the alkali-denatured serum was loaded per well of the minifold slot-blot apparatus and the solution was passed through the membrane applying a water-suction pump. The membranes were removed from the slot-blot apparatus and neutralized by floating 5 min on a solution of Na-acetate, 3M; pH 5.0/25 °C and 2 x 5 min on a solution of 2 x SSC.The filters were air dried and baked 2 h at 80 °C in a vacuum oven.
3.1) Fixing of suspension cells Suspension cells (B95-8 and BJAB) were washed in PBS (phosphate buffered saline: Na/K-phosphate, lOmM; KC1,2.7mM; NaCl, 120mM; pH 7.2/25 °C) after the fourth day of splitting and dropped on precoated slides. B95-8 cells were derived from a marmoset B-lymphoid cell line immortalized by EBV. Each individual B95-8 cell contains a few EBV genomes per cell with restricted expression of viral genes. 5-10% of the cells spontaneously enter a lytic cycle of viral replication; these cells contain up to 10000 viral genomes per cell. Degreased slides were baked for l h at 100 °C, incubated for 3 h at 65 °C in 3 x SSC; ficoll, 0.02% (w/v); polyvinylpyrrolidone, 0.02% (w/v); bovine serum albumin, 0.02% (w/v), dipped shortly in H2O dest. and fixed in 3:1 (v/v) ethanol/acetic acid for 20 min at 22 °C.The size of the cell spot fitted under an 18 x 18 mm coverslip.The airdried cells on these slides were fixed for 2.5 min at 22 °C in freshly prepared paraformaldehyde, 4% (w/v) in PBS. 3.2) Preparation of digoxigenin-labeled probe 26 different EBV[£amHI]-fragments (not the EBV[BamHl]Kfragment due to cross-reaction with cellular DNA) were eluted from agarose gel after restriction enzyme digestion and electrophoresis, pooled and labeled with digoxigenin as described in Materials and Methods, section A.2.
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R. Seibiet al.
3.3) In situ hybridization Protocols were derived from Wolf et al.'16', Falser et al.' 17 ' and Heiles et al.'10' with some modifications. HC1 treatment as suggested by some authors was omitted, as it seemed to introduce false positive signals in some cases. The slides were washed twice for 10 min at 22 °C in PBS; MgCI2, 5mM, and incubated at 50 °C for 30 min in 2 χ SSC; EDTA, 5mM. After a 15 min treatment with 1 μ-g proteinase K/m/ PBS at 37 °C, the slides were washed for 10 min at 22 °C in PBS containing glycine, 0.02% (w/v), followed by post-fixation for 20 min at 22 °C with freshly prepared paraformaldehyde, 4% (w/v). Final washing was twice for 15 min at 22 °C with PBS; MgCl 2 ,5mM. Pre-hybridization was carried out for 15 min at 22 °C in 50 μ//5ροΙ of 6 x SSC; formamide, 45% (v/v); 5 x Denhardt's solution; heat-denatured salmon sperm DNA, 100 pg/ml. After careful removal of the pre-hybridization solution without washing 20 μ/ of probe-hybridization solution were placed under a siliconized coverslip containing 7.5 ng digoxigenin-labeled probe in 6 x SSC; formamide, 45% (v/v); 5 x Denhardt's solution; dextran sulfate, 10% (w/v). The edges were sealed with rubber cement (Fixogum, Marabu). The cellular and probe DNAs were denatured while placing the slides directly on a hot plate (Ortho Diagnostics) at 90-94 °C for 5 min. The slides were cooled on ice and incubated at 42 °C for 16 h. After the coverslips had been removed the slides were washed twice at 42 °C for 15 min in 6 x SSC; formamide, 45% (v/v), then twice at 22 °C for 5 min in 2 x SSC, and finally twice at 50 °C for 15 min in 0.2 x SSC. 3.4) Detection The detection reaction was carried out as described in Materials and Methods, section A.4.The colour reaction was stopped after 2-6 h withTris/HCl, 10mM; EDTA, ImM; pH 8.0/25 °C. No further staining was performed on the slides, they were evaluated with a differential interference contrast device (Zeiss).
E) In situ hybridization ofmetaphase chromosomes Cells were exposed to colcemid, 0.05 yug/m/, for l h before fixation. Chromosome preparation was performed as described in ref.'18'. In situ hybridization was performed according to the protocol described by Ambros et al.1191. To minimize non-specific binding of the probe, slides were treated with 100 μΐ of DNase-free RNase (100 /Ag/m/, Boehringer Mannheim) and sealed with a coverslip. After 1 h incubation at 37 °C, coverslips were removed and the slides were washed with water and dehydrated with alcohol. 50 μ,/ hybridization solution was placed on the slides under a coverslip, sealed with rubber cement, then chromosomes and the probe were denatured at 72-74 °C for 10 min. The hybridization solution was composed of 2 x SSC; formamide, 50% (v/v); dextran sulphate, 10% (w/v); sonicated salmon sperm DNA, 0.5 mg/m/; digoxigeninlabeled probe, 16 ng/m/. Hybridization was performed at 39 °C for 14 h. Nonspecific binding of labeled probe was avoided by appropriate hybridization conditions and by additional washing steps at a temperature adequate to remove adventiously bound probe. After removing the coverslips the slides were either washed at room temperature for 5 min with 2 x SSC or at 39-41 °C for 10-30 min with 2 x SSC; formamide, 50% (v/v), or alternatively three times at 37 °C for 5 min with 2 x SSC, one for 3 min with PBS;Triton X-100, 0.1% (v/v), and again three times for 5 min with PBS. For detection of hybridized digoxigenin-labeled probe the slides were washed withTris/HCl, lOOmM; NaCl, 150mM; pH 7.5/25 °C and incubated for 1 h at room temperature with diluted (Dig):CIAP-solution (150 mU/m/; Boehringer Mannheim). The slides were subsequently washed with Tris/HCl, lOOmM; NaCl, ISOmM; pH 7.5/25 °C, and then with Tris/HCl, lOOmM; NaCl, lOOmM; MgCl2, 50mM; pH 9.5/25 °C. Colour development was stopped after 1 h by washing the slides extensively in Tris/HCl, lOmM; EDTA, ImM; pH 8.0/25 °C. For microscopic analysis a Leitz
Vol. 371 (1990))
photomicroscope equipped with reflection-constrast'20' was used. Photographs were taken on Kodak Ektachrome andTechnical Pain film. F) Labeling of nucleic acids during polymerase chain reaction Serial dilutions of 10 ng to 0.1 pg from linearized plasmid pSPTlSrteo containing the neomycin gene (940 bp) were used a:s templates in polymerase chain reactions according to Saiki et al.'21j'. These plasmid concentrations correspond to 2.3 ng to 23 fg o>f neomycin-specific insert sequences. The PCR was primed with SP
