solution of 'olefin' 1 [10 mg/100 ml n-hexane (UVasol) or methanol] for 20 s. ..... l aliquots of labelled oligonucleotide containing 50, 10, 2, 0.4 and 0.08 fmol/l.
. 1995 Oxford University Press
Nucleic Acids Research, 1995, Vol. 23, No. 22 4657-4663
Non-radioactive detection of oligonucleotide probes by photochemical amplification of dioxetanes Frank Schubert*, Andrea Knaf, Uwe Moller and Dieter Cech Humboldt-Universitat zu Berlin, Institut fOr Chemie, Hessische Strasse 1-2, 10099 Berlin, Germany Received August 4, 1995; Revised and Accepted October 13, 1995
ABSTRACT We describe a new method of non-radioactive labelling and detection of oligonucleotide probes. The approach is based on a simple chemical principle. Oligonucleotides labelled with methylene blue (a photosensitizer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2-[3-(hydroxyphenyl)methoxymethylene] adamantane is added to the membrane and the membrane is irradiated with a tungsten lamp light source through a cut-off filter. Thermally stable dioxetanes are amplified during irradiation at the positions of the labelled probe. These amplified dioxetanes are detected using chemically triggered chemiluminescent decay. Signals are recorded on commercial X-ray film. Detection is possible immediately after the last washing step and a hard copy of the blot is obtained within I h. Dependent on the level of the target sequences, the sensitivity of the method allows detection of 0.3 pg single-stranded M13mpl8(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunological reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physical detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The synthesis of the olefin, conditions of hybridization and the protocol of detection are described in detail.
INTRODUCTION Technologies using labelled oligonucleotide probes have provided a powerful tool to solve problems in DNA analysis. This technique now has a variety of applications, from medical diagnostics and forensic analysis (analysing biological evidence of crimes) to the identification of certain DNA fragments in agriculture or basic research (1-2). Since the introduction of this technology in the mid 1970s the use of radioactively labelled probes has limited its widespread application, due to the technical requirements in using radioactive labelling reagents such as 32p or 5S. Additionally, concerns over safe working practices with radioisotopes, health risks, hidden costs and the low stability of radioactively labelled probes are features that have hampered the *
To whom correspondence should be addressed
widespread adoption of this technology in applied settings. Progress in the methodology of using oligonucleotide probes for DNA analysis as an everyday technology has culminated in the development of non-radioactive systems. Recent advances in substrate chemistry and probe labelling have dramatically increased the sensitivity of non-radioactive methods and, consequently, different sensitive non-radioactive systems have been published, some of which are commercially available today (3-5). Mansfield et al. recently published a comprehensive article on the state of the art of non-radioactive DNA labelling and detection systems (6). The identification of any immobilized target DNA (e.g. blotted DNA) by non-radioactive probes is mainly based on two detection principles, namely biochemical or physical approaches. Biochemical approaches indirectly detect reporter molecules using products of an enzymatic reaction. The enzymes, usually alkaline phosphatase (7) or horseradish peroxidase (8), are attached to the probes either directly (9) by covalent cross-linking reagents or indirectly using affinity ligands. Biotin (10), haptens (11) (e.g. digoxigenin, fluorescein and a-DNA), enzyme inhibitors (12) or platinum complexes (13) have been described in this context. Signal generation is accomplished through formation of fluorescent (14) or coloured (15) precipitates, but triggering of chemiluminescence by marker enzymes (16) is the most important detection principle in non-radioactive systems of DNA detection based on biochemical reactions. In physical approaches the reporter molecules can be detected directly. Most systems use the fluorescent properties (both static and dynamic fluorescence measurements) of fluorophores such as fluorescent dyes (17) or transition metal complexes (18). Electrochemically generated chemiluminescence of ruthenium complexes has also been used (19). However, except for hydrolysis of acridinium esters with hydrogen peroxide under alkaline conditions (20), simple chemical detection reactions have not yet been described. Chemical detection has lagged well behind biochemical or physical approaches, largely because of unknown pure chemical reactions in which signal amplification is influenced by one component at very low concentrations (a few amol). In view of the simplicity of chemical reactions in contrast to, for example, enzymatic reactions, concepts using signal amplification by chemical means may have an advantage over existing methods. Thus we were encouraged to search for useful chemical complements to the enzymatic cleavage of dioxetanes, which is accompanied by emission of light. The detection of this chemiluminescence requires little in terms of equipment and, moreover, the generation of signals is relatively fast (ranging from minutes to overnight exposure of X-ray film).
4658 Nucleic Acids Research, 1995, Vol. 23, No. 22 0 o pOCH31 R
101
I
R - P03>, CH3CO
+
*0
Light at 470 nm
Figure 1. Chemiluminescent decomposition of thennally stable dioxetanes after chemical/enzymatic formation of a thermally unstable phenolate anion.
Therefore, it is the method of choice in non-radioactive detection systems (21). The basic mechanism of the commercially available DIG-System is outlined in Figure 1. Thermally stable dioxetanes are the most favoured reagents used for the generation of chemiluminescence by enzyme-labelled DNA and oligonucleotide probes. Enzymatic cleavage of the phosphate ester of disodium-3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPDTM) drastically diminishes the thermal stability of the dioxetane ring and, consequently, the dioxetane decomposes and light is emitted. From the chemical point of view these dioxetanes can be easily formed by treatment of suitable olefms with singlet oxygen (102). Thus the herein described method has conceptually the idea that a 102 photosensitizer covalenfly attached to the oligonucleotide probe should be capable of forming intermediates of dioxetanes in the presence of suitable olefms. The procedure has the advantage of bringing the chemical reaction direct to the hybridization zone where the labelled probe initiates a photochemical cascade. The idea was stimulated by our recently published results on covalent coupling of methylene blue to oligonucleotides (22). Methylene blue [3,7-bis-(dimethylamino)phenazathionium chloride] is a well-known 102 photosensitizer. Hence, oligonucleotides labelled with methylene blue should be able to amplify thermally stable dioxetanes based on repeated excitation/oxygen quenching cycles (Fig. 2). Unlike procedures using enzymatic decomposition of AMPPDTm, the most important feature of the described procedure is to apply olefins delivering dioxetane structures which are decomposed by a simple change of pH. Thus the procedure is not complicated to perform. The major advantages of our method include speed and efficiency. For the first time the application of methylene blue as a photosensitizer for analysing target sequences of immobilized DNA is described using its ability to amplify thermally stable dioxetanes.
MATERIALS AND METHODS 5'-Amino-functionalized oligonucleotides were synthesized using synthons obined from Phmacia (C-, T-, G-, A- and TFA aminolinker amidites) and Glen Research (5'-amino modifier C3 and 5'-amino modifier C12) on a Gene Assembler® DNA synthesizer (Pharmacia). M13mpl8(+)ss plasmid DNA was purchased from Pharmacia. All other chemicals were obtained from Aldrich. High performance luminescence detection film (HYPERfilmTM-ECL) was purchased from Amersham. Dot blot and Southern blot hybridizations were performed on neutral HybondNTm membranes (Amersham). The TurboblotterTm system from Schleicher & Schuell was used for Southem blot transfer according to the instructions of the supplier. The 'H-NMR spectra were measured at 300 MHz using a Bruker AMX 300 spectrometer. Chemical shifts for IH are given in p.p.m. (6) relative to tetramethylsilane. Melting profiles were recorded with a Cary- 1 UV/Vis spectrophotometer connected to a thermocontroller (Varian, Australia). The hybridization oven OV-1 was kindly provided by Biometa, Germany.
Oligonucleotide labelling Synthesis of the modified methylene blue and coupling of carboxy modified methylene blue to 5'-amino-functionalized oligonucleotides were performed as previously described (22). For that, 1.9 OD of aminoalkylated oligonucleotide (6.3 nmol) and 63.4 jg activated dye (126 nmol) were incubated in 0.1 M potassium dihydrogen phosphate, pH 8, DMF (1:2 v/v, total volume 300 gl) overnight in the dark at room temperature. After evaporation of the solvent the product was redissolved in bidistilled water (100 ,ul) and successively treated with n-butanol (1 ml). The product was collected by centrifugation, repeatedly extracted with n-butanol and dried. The dried product was stored in the dark to avoid undesired photodestruction of the DNA.
Synthesis of 2-[3-(hydroxyphenyl)methoxymethyleneJaamantane (1) In a 500 ml three neck flask fitted with a reflux condenser, an additional funnel and connected to a nitrogen line, 22.1 ml titanium tetrachloride (201 mmol) were added dropwise over 30 min to 230 ml ice cold dry THF, followed by portionwise addition of 5.73 g lithium aluminium hydride (151 mmol) under rigorous stirring. After removing the ice bath 15.7 ml triethylamine (113 mmol) were added and the suspension was refluxed for 1 h. A solution of 3.8 g 2-adamantanon (25.3 mmol) and 6.29 g methyl-3-t-butyldimethylsilyloxybenzoate (23.6 mmol) in 40 ml absolute THF was added dropwise to the refluxing mixture over 45 min. Refluxing was continued for an additional 1.5 h. After cooling to room temperature the suspension was diluted with 200 ml diethylether and continously stirred for 10 min. The precipitate was filtered off and the filtrate diluted with 100 ml water. The organic layer was separated, dried over anhydrous sodium sulphate, filtered and evaporated under vacuum to dryness. Chromatography on silica gel with toluene as eluent yielded 1.75 g (27%)
2'-[(3-t-butyldimethylsilyloxyphenyl)methoxymethylen]tricyclo(3.3.1.1.3'7)decane as an oil. 'H-NMR (CDC13) 6 (p.p.m.): 0.19 (s, 6H, H-9); 0.98 (s, 9H, H- I 1); 1.77-1.96 (m, 12H, H-adamantyl); 2.63 (s, 1 H,
Nucleic Acids Research, 1995, Vol. 23, No. 22 4659 'Photosensitizer + hv Oligonuckotide __--_
3[Photosensitizer]*
* [Photosensitizer] -f--Olionudeotide
|
3rphntancpcitiyffrl* V'JIUMMseUMU--
+ Tr
I
IOligonudeotide I
3f_ _ %2-
Flgoudeoi]
Photodestruction
OCH3
10
-
OH
I
Figure 2. Photochemical amplification of thermally stable dioxetanes via excitation/deactivation cycles of photosensitizers.
H-adamantyl); 3.24 (s, 1H, H-adamantyl); 3.28 (s, 3H, OCH3); 6.74 (m, 2H, H-2 and H-4); 6.91 (dt, lH, H-6); 7,19 (t, lH, H-5). The obtained 2'-[(3-t-butyldimethylsilyloxyphenyl)methoxymethylen]tricyclo-(3.3.1.1.3,7)decane (500 mg, 1.3 mmol) was dissolved in absolute THF. An aliquot of 1.5 ml 1.1 M tetrabutylammoniumfluoride in THF was added and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC on silica gel developed in hexane/ethyl acetate (9: 1). When the reaction had gone to completion the mixture was transferred to a separating funnel, diluted with diethylether and then washed three times with water. The aqueous layer was washed twice with freshly distilled diethylether. The combined organic phases were dried over anhydrous sodium sulphate, filtered and evaporated under reduced pressure. Yield: 323 mg (92%) of 1. IH-NMR (CDCl3): 6 (p.p.m.) 1.76-1.94 (m, 12H, H-adamantyl); 2.64 (s, 1H, H-adamantyl); 3.22 (s, 1H, H-adamantyl); 3.32 (s, 3H, OCH3); 6.02 (s, 1H, OH); 6.8 1(m, 2H, H-4 and H-6); 6.86 (s, 1H, H-2); 7.19 (dd, 1H, H-5).
Blotting Dot blot. The target DNA [Ml3mpl 8(+)ss] was diluted in DNA dilution buffer (50 ,ug/ml salmon sperm DNA in a 10 mmol solution of Tris-HCl, pH 8, containing 1 mmol EDTA). Final concentrations were 100, 50, 25, 10, 5, 1, 0.5 and 0.1 ng/,l respectively. After denaturation of the solutions in a boiling water bath for 10 min 1 gl denaturated DNA was transferred to a sheet of Hybond-NTm nylon membrane and baked at 1200C for 0.5 h.
Southern blot. After electrophoresis of the M13mpl8(+)ss plasmid DNA in a 0.7% agarose gel the DNA was transferred by the TurboblotterTm system. The neutral transfer was performed as follows: (i) the gel was shaken in denaturing buffer (0.5 M NaOH, 1.5 M NaCl) for 30 min at room temperature; (ii) the gel was rinsed with distilled water and transferred to neutralizing buffer (0.5 Tris-HCl, pH 7.0,1.5 M NaCl, 30 min at room temperature); (iii) the gel was treated with transfer buffer (20x SSC, pH 7) for 30 min; (iv) the gel was applied to a Hybond-Nrm nylon membrane for 3 h; (v) the membrane was washed with 2x SSC for 5 min and, finally, baked at 120'C for 0.5 h.
Hybridization Hybridization was carried out without blocking reagents, such as Denhardt's solution, or heterologous DNA. Prehybridization was performed for 30 min at 45°C in a buffer containing 0.25 sodium phosphate, pH 7.2, 7% SDS. After addition of the methylene blue-labelled oligonucleotide (10 pmol/cm2) the hybridization was run overnight (16 h) at 45 °C in the dark. The membranes were hybridized under constant rotation in a thermoregulated hybridization oven. The buffer volumes were 1 mI/cm2 membrane. After hybridization the membrane was washed with the following buffers: 15 min in 6x SSC at room temperature, 5 min in 3x SSC at 45°C, 5 min in 3x SSC and twice for 5 min in hybidization buffer at room temperature. The membrane was then allowed to dry at room temperature.
Detection The trigger solution was prepared by addition of a 40% aqueous solution of tetrabutyl ammonium hydroxide (TBAOH) to water untill pH 13 was reached. The dry membrane was dipped in a solution of 'olefin' 1 [10 mg/100 ml n-hexane (UVasol) or methanol] for 20 s. After drying the membrane was placed on a black non-reflecting background and illuminated with a 100-150 W tungsten light source for 10 min. A coloured glass filter (cut-off wavelength 620 nm) was located between the light source and the membrane. The illuminated side of the membrane was placed on an X-ray film coated with food wrap. Finally, the membrane was soaked with the trigger solution (TBAOH, pH 13) and the film exposed for 1 h.
RESULTS AND DISCUSSION Our labelling and detection strategy took account of two factors: we were interested in developing a chemical route to a thermally stable dioxetane intermediate being formed at the position of a labelled DNA or oligonucleotide probe from a suitable olefin by treatment with photochemically generated singlet oxygen. Since we intended to use an olefin that fulfilled the above requirments, it was necessary to find a suitable photosensitizer. The latter should initiate an amplification mechanism and, therefore, be
4660 Nucleic Acids Research, 1995, Vol. 23, No. 22 sufficient to generate a detectable signal in very slight quantities. The mechanism of amplification is outlined in Figure 2. Accordingly, we first devised the synthesis of an olefmnic structure which generates thermally stable dioxetanes. Dioxetanes are generally synthesized by introduction of photochemically generated singlet oxygen (102) into olefinic double bonds (23). The analytical application of that type of reaction of olefins with singlet oxygen in combination with both decomposition of the dioxetanes formed and detection of the emitted light was suggested by Shellum et al. (24) for HPLC analysis of polycondensed aromatics. McCapra et al. (25) suggested a similar scheme for DNA detection. The principal obstacle to its application rests in decomposition ofthe dioxetanes used. Decomposition has to be carried out under forcing conditions of temperature (150°C) and requires additional equipment for the detection of light at these temperatures. Thus the procedure is very expensive and complicated, which is a disadvantage for wide application. It was, therefore, of interest to prepare an olefin with a subtle modification, in the hope that the thermal stability of the amplified dioxetane might be easily abolished by a simple chemical reaction. 2-[3-(Hydroxyphenyl)methoxymethylene]adamantane (compound 1, Fig. 2) proved suitable for this reaction. The chemical synthesis of 1 was carried out via a modified McMurry reaction (26) with 2-adamantanone and the 3-t-butyl-dimethylsilylether of 3-hydroxybenzoic acid methyl ester as starting materials. Low valency titanium was used as the catalyst. Deprotection of the silylether using tetrabutylammonium fluoride led to 1 in satisfactory yields (see Materials and Methods). The structure assigned to 1 was confirmed by IH-NMR spectroscopy. The spectrometric data for 1 prepared in this way are identical to the described data (27). This olefin reacts easily with photochemically generated singlet oxygen to give the corresponding 3-[2-spiroadamantane-4-methoxy-4-(3'-hydroxyphenyl)]- 1,2-dioxetane 2. We have investigated this reaction by HPLC. 2 was the only product obtained and side reactions caused by photodestruction were not observed (data not shown). The structure of isolated 2 was confimed by both 1H- and 13C-NMR spectroscopy. After deprotonation of the phenolic hydroxyl group 2 loses its thermal stability and its decay is accompanied by the emission of light (max 470 nm). According to Figure 2 the required 102 is produced by energy transfer from an electronically excited molecule of the dye. The dye reaches its ground state during energy transfer and is accessible to a new activation/deactivation cycle. Therefore, a very small amount of the photosensitizer is sufficient to produce a relatively large amount of dioxetane via 102 trapping. Thus a
photosensitizer had to be found which is capable of generating 02 in trace amounts and yet yielding enough detectable dioxetane. For this purpose three organic dyes, erythrosine B, rose bengal and methylene blue, were studied as potential photosensitizers. All of them have been extensively studied as potential generators of singlet oxygen, due to their high quantum yield of electronic intersystem crossing (ISC). Hence, to test this possibility we started by comparing the detectable amounts of the dyes in the presence of 1 (Table 1). For that, series of different concentrations of each dye were spotted onto nylon membranes normally used in biological blotting experiments. After drying the membranes were dipped in a solution of 1 and dried again. Finally, the membranes were irradiated with a tungsten lamp through an appropriate cut-off filter (Xcut-off 530 nm for erythrosine B and rose bengal; Xcut-off 620 nm for methylene blue) for -10 min and successively placed on a wrapped sheet of X-ray film. Emission of light was triggered by covering the membrane with filter paper soaked with a solution of TBAOH, pH 13. Film exposure was for 30 min and, finally, the film was developed as usual. The results of these experiments are listed in Table 1. Additionally, some photochemical properties of the three dyes used are given in Table 1. Under the present experimental conditions methylene blue was the best sensitizer, due to its producing detectable amounts in the amol range (10-18 mol). The achieved sensitivity is sufficient to localize a single copy gene in -1 ig human genomic DNA. To achieve the maximum sensitivity both the influence of irradiation time of the membrane and of olefin concentration on the membrane were studied. Best results were obtained with irradiation of the membrane for 10 min. Prolonged irradiation for 20, 40 and 60 min did not improve the strength of the signal; the sensitizer was completely destroyed during the first minutes due to undesired side rections. This may be useful, especially for reprobing the same membrane. Furthermore, it was found that a concentration of the olefm of 0.1 mg/ml solvent led to the best results. Lower amounts of olefin caused weaker signals on the X-ray film. In contrast, higher amounts of olefin (up to 1 mg/ml solvent) did not result in a stronger signal or improved sensitivity. Taken together the results suggest that we have found an analytical principle based on a pure chemical reaction which allows the detection of photosensitizers in the amol range. This process is depicted in an idealized form in Figure 3. The principal strenghts of this method relative to existing methods ofDNA analysis are in the elimination of: (i) any biological element during the process; (ii) time-consuming enzymatic decomposition of the dioxetanes; (iii) expensive equipment and chemicals; (iv) the use of radioisotopes.
Table 1. Spectroscopic data and detectable amounts of photosensitizers used in developement of the non-radioactive labelling and detection system based on amplification of thermally stable dioxetanes
X max (nm)
Erythrosin B Rose bengal Methylene blue
530 (MeOH) 560 (MeOH) 668 (MeOH)
The spectroscopic data were taken from (32). aA filter with .cut-off 530 nm was used.
£ (l/mol/cm) 94 000 104 000 89 000
*
('02)
0.62 0.76 0.23
Detectable amount (mol) I0-16a
10-16a 1-418
Nucleic Acids Research, 1995, Vol. 23, No. 22 4661
7
/
/
/ /
/.-.4. */ deposition of olefin I
immobilized target DNA and oligonulceotide probe labelled with the photosensitizer
Z:
/ /t Z. / .-
irradiation at 670 mun ii (a)
009
*.
exposure to X-ray film at pH 13 ill
0CH3
/ /
/ Figure 3. Basic principle of the non-radioactive detection of target DNA using oligonucleotide probes tagged with photosensitzers. A suitable olefin is applied to the membrane (i). During irradiation with light [ii(a)] the sensitizer leads to production of singlet oxygen. Thermally stable dioxetane is amplified exclusively at the position of the oligonucleotide probe [ii(b)]. Chemical formation of the phenolate ion on X-ray film (iii) leads to light emission and produces signals on the film.
Further efforts to evaluate the analytical potential of this chemical system of amplification were directed toward labelling of oligonucleotides with methylene blue and refining our recently published procedure for coupling methylene blue to oligonucleotides (22,28). Owing to the absence of any functional group within the methylene blue molecule complications arose with covalent attachment to oligonucleotides. The solution to this problem was to synthesize a methylene blue derivative bearing an alkyl chain with a terminal carboxylate residue. In a previous paper (22) we reported that modified methylene blue derivatives can be obtained according to procedures described by Motsenbocker et al. (29). Through activation by the N-hydroxysuccinimido ester approach the terminal carboxy group permits coupling of the dye to the S'-amino modified oligonucleotide currently also used for labelling oligonucleotides with digoxigenin or biotin. Using freshly activated ester the yields of the labelling reaction can be increased to >90% labelled oligonucleotide. Thus HPLC purification of the labelled probes is not necessary. Oligonucleotide probes were simply isolated by either precipitation or extraction of the reaction mixture with n-butanol. In order to study the influence of the modification on methylene blue-oligonucleotide conjugates, binding properties with complementary oligonucleotides were evaluated. Melting temperatures (Tm) of duplexes formed by the entirely modified 20mer conjugate 5'-MB d(CC AGG GTT TTC CCA GTC ACG) with d(GG TCC CAA AAG GGT CAG TGC) and a 33mer d(CATTGC GG TCC CAA AAG GGT CAG TGC TGCAACA) were 70.5 and 70.7°C, which are 2°C higher than for the unmodified counterparts (Tm values of the unmodified duplexes were 68.5 'C for the 20mer and 67.2°C for the 33mer respectively).
These results indicate that interactions between the methylene blue-labelled probe and 5' overlapping DNA or oligonucleotide fragments influence neither the stability of the duplex nor the hybridization behaviour of the conjugate with complementary strands in an undesired way. The small increase in melting temperature is possibly due to weak electrostatic interactions between the positively charged dye and complementary strands. Interaction of methylene blue itself and DNA strands has been extensively investigated and reported (30). Methylene blue is able to intercalate into DNA strands and electrostatic forces have also been described. Due to the only weak increase in the Tm value between methylene blue-oligonucleotide conjugates and their complementary counterparts we favour the electrostatic influence. Moreover, the absorption maximum of methylene blue did not show any change in oligonucleotide duplexes. A bathochrome shift of the absorption maximum of the dye, a result of intercalation and the resulting stacking interaction between free methylene blue and polynucleotides, was not observed. Taken together, the results suggest that duplexes formed by labelled oligonucleotides with DNA targets are stable and so we conclude that the labelled oligonucleotides should be useful as sequence-specific probes. Finally, studies of the application of methylene blue-oligonucleotide conjugates were performed on a model system of commercially available DNA (the principal path is shown in Fig. 3). In order to detect target DNA via amplification of thermally stable dioxetanes target DNA was immobilized on a nylon membrane. After hybridization with a methylene blue-labelled oligonucleotide probe and successive stringency washing steps the membrane was impregnated with the corre-
4662 Nucleic Acids Research, 1995, Vol. 23, No. 22 0--0fmul
_
-
ti.>0 i ,;
10 fmol I
2o2 fmol an
10.4 fmol
i+*
I.
Z
as-
(0.08 fnmol
Figure 4. Detection limit of a methylene blue-labelled oligonucleotide dotted onto Amersham Hybond NTM membrane. Detection was performed as described in Materials and Methods. Oligonucleotide at 0.08 fmol was detected after 30 min exposure on a X-ray film.
sponding olefin in only a few seconds. Subsequent drying and irradiation of the membrane by a simple commercially available tungsten lamp using a corresponding cut-off filter led to amplification of the dioxetanes at the probe position on the membrane. Signal generation was achieved by alkaline initiation of dioxetane decay, which is accompanied by the emission of light. The mode of detection is most similar to those of the radioactive approach and detection is possible almost immediately after the washing steps. Any additional blocking operations or binding of affinity ligands are not necessary. To investigate the upper concentration limit for detection of oligonucleotide probes we firstly analysed labelled probes in the absense of target DNA. For that, 1 ,l aliquots of labelled oligonucleotide containing 50, 10, 2, 0.4 and 0.08 fmol/l respectively were spotted onto a nylon membrane (either onto a neutral or a positively charged membrane). The labelled oligonucleotide was fixed by baking the membrane (120°C, 0.5 h) and subjected to the above described procedure (Fig. 4). Figure 4 shows that we detected 0.08 fmol or 80 amol labelled oligonucleotide after a 30 min exposure. The achieved sensitivity is similar to that obtained using other non-radioactive systems, especially the DIG/Anti-DIG-aPho system (31). With its short exposure time the new method is somewhat of an improvment over the DIG system, where the same sensitivity is achieved after exposure overnight. Taking the above results on hybridization and sensivity of detection of oligonucleotides labelled with methylene blue together, it was particularly interesting to confirm the method on a target DNA sequence. For this purpose, we used single-strand M13mpl8(+) as the immobilized target and the 20mer oligonucleotide probe 5'-MB-d(CC AGG GTIT TTC CCA GTC ACG)-3', complementary to the lacZ region ofthe target. First, in a dot blot experiment 1 pl of a series of dilutions containing 100, 50, 25, 10 1, 0.5 and 0.1 ng respectively of the target were dotted on a nylon membrane and fixed by baking the membrane at 120°C for 30 min. Hybridization was performed overnight at 45°C in a universal hybridization buffer containing 7% SDS and 0.25 M disodium hydrogen phosphate, pH 7.2. The buffer is particularly recommended because it does not contain any components of protein. Proteins as components of the buffer may interact with the label, preventinging optimal hybridization. In order to detect the immobilized target DNA the membrane was washed, dried and, finally, soaked with the solution of olefin. No steps are time-consuming and all can be performed in only a few minutes. Figure 5 shows the processed membrane after 10 min
*' .t
rig
Figure 5. Detection of single-stranded M13mpl8 DNA in dot blot format via amplification of dioxetanes using a methylene blue-oligonucleotide conjugate. The amount of DNA detected is dependent on the level of target sequence.
irridiation. It demonstrates that the amount of dioxetane produced is sufficient to localize -0.1 ng plasmid DNA (or -0.3 pg target sequence). Finally, we have studied the detection of point mutations in a HLA oligonucleotide hybridization assay. An oligonucleotide probe [MB-d(TAA GTT TGA ATG TCA TITIT)] complementary to the HLA-DRB 1 gene of chromosome 6 was synthesized and labelled with methylene blue. Four positive DNAs from different forensic samples and four negative controls were amplified using PCR and analysed as described in the dot blot experiment. All four positive samples were unambiguously detected with the dioxetane amplification system. Parallel studies were performed with digoxigenin-labelled oligonucleotides. Neither sensitivity nor specifity differences could be found. In an analogous experiment the method was tested in a Southern blot. DNA amounts mentioned in the description of the dot blot experiments were electrophoresed in a 0.7% agarose gel and transferred to a neutral or positively charged nylon membrane using the TurboblottereM system. Immobilization, hybridization and detection were performed as desribed in the dot-blot experiments. Ml3mpl8 DNA was detected at a concentration of 25 ng in this way, which correspond to 75 pg target sequence
CONCLUSIONS The presented method shows a simple and efficient way of analysing DNA targets in blotting experiments by a non-radioactive approach. We used the kinetics of light emission of dioxetanes on nylon membranes. To amplify the dioxetanes we used a photosensitizing process involving oligonucleotide probes labelled with methylene blue, which is a very efficient generator of singlet oxygen. The singlet oxygen initiates transformation of a suitable olefin applied to the membrane into a dioxetane, which is simply decomposed by changing the pH. Measuring the emitted light in a multiple film exposure is possible, both to detect small amounts of DNA and to differentiate between strong signals (a relatively short exposure) and rather weak signals (a long exposure). The approach does not require any additional blocking reagents or washing operations. Furthermore, expensive apparatus is not necessary. The generation of signals is possible immediately after hybridization and stringency washing. Thus the method described combines both the advantages of radioactive labelling methods, e.g. easy labelling procedures and stability of the label during handling of the probe, and non-radioactive systems, e.g. safety of handling, probe stability and sensitivity. No additional blocking operations or binding of affinity ligands are
Nucleic Acids Research, 1995, Vol. 23, No. 22 4663 Additionally, the label is a simple small molecule which is stable under different conditions used for DNA denaturation, hybridization and other operations in molecular biology (e.g. PCR techniques). The simplicity, speed and convenience of the method suggest that it can prove very useful in routine analyses that requires small quantities of DNA. Further improvements in the sensitivity of the system may be achieved by introduction of nucleoside triphosphates labelled with methylene blue. This may open up the possibility of multiple labelling of DNA or oligonucleotides and allow procedures such as random primed DNA labelling or the use of terminal transferase for multiple incorperations of the photosensitizer. Further research is being done in this area at present. necessary.
ACKNOWLEDGEMENTS We are grateful to Prof. Adam and his group from the University of Wurzburg for a helpful introduction to the McMurry reaction and dioxetane chemistry. The authors thank Dr M. Nagy of the Institut fur Gerichtliche Medizin der Humboldt-Universitait for providing the clinical samples of the HLA oligonucleotide typing. We are indebted to Dr S. Schmidt for critical reading and discussion of the manuscript. This work was supported by the Bundesministerium fur Bildung und Forschung, 0310 255A.
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