Improved Synthesis of DBM Paper - Science Direct

ANALYTICAL

120, 259-261 (1982)

BIOCHEMISTRY

Improved

Synthesis

of DBM Paper

D. CHRISTOPHE, H. BROCAS, ANDG. VASSART Institut

de Recherche

Interdisciplinaire, Rue Evers,

Universith Libre 2. 1000 Brussels,

de Bruxelles, Belgium

Faculte’

de Midecine.

Received July 16, 198 1 A modified synthesis of DBM paper is described which is simple and easily reproducible. The method previously published has been improved in order to optimize the reaction conditions of each step. The paper obtained in this way shows a very high binding capacity for denatured DNA.

Since its first use by Alwine et al. (1,2), DBM paper has proved to be a very useful tool for the study of nucleic acids by hybridization methods. Although nitrocellulose is frequently used for blotting experiments, DBM paper is required when small fragments of DNA or RNA are to be trapped, since it binds them covalently. This bond between DBM paper and single-stranded nucleic acid allows the melting of the doublestranded structure formed after hybridization without releasing the strand fixed on the paper. This advantage over other methods can be exploited, e.g., for the characterization of cDNA sequences inserted in recombinant plasmids by the hybridization-elution-translation technique. We find, however, that the procedure described by Alwine et al. (1,2) for the synthesis of DBM paper has proved to be of poor reliability, especially where it concerns the binding capacity of the paper. The same problems have been encountered by other laboratories also (3). Therefore, we describe here a modified synthesis- of DBM paper which is easily reproducible. The procedure is simple and leads to a paper having a very high binding capacity. MATERIALS

AND METHODS

N - (3 - Nitrobenzyloxymethyl)pyridinium chloride (NBPC)’ was purchased from BDH ’ Abbreviations

Biochemicals, Poole, United Kingdom, and N,N-dimethylformamide (DMF) for spectroscopic use (UVASOL) from E. Merck, Darmstadt, West Germany. Synthesis of ABM paper. Two hundred eighty milligrams of NBPC is added to 1.2 ml of DMF in a stoppered vial and this suspension is heated in a water bath (40°C) until a turbid solution is obtained. A circle (9 cm diameter) of Whatman 540 filter paper is soaked with the solution and immediately placed in an oven at 130-135°C for 30 min. After cooling, the paper is washed twice with 50 ml acetone and twice with 100 ml water. It is then agitated in a freshly prepared solution of 10 g sodium dithionite in 100 ml of 50 mM sodium hydroxide for 2 h at room temperature. After four washes with water ( 100 ml/wash) and two acetone washes (50 ml/wash), the ABM paper is oven dried (10 min at 37°C) and stored at -20°C. Fixation of DNA to DBM paper. The desired area of ABM paper is cut for the diazotization reaction. For each square centimeter of ABM paper, 1 ml of 1 M hydrochloric acid and 22 ~1 of a freshly prepared aqueous solution of sodium nitrite (50 mg/ml) are used. Vessels and solutions are cooled to 0°C before use. After 30 min at 0°C the DBM paper is washed three times methyl)pyridium chloride; DMF, N,N-dimethylformamide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

used: NBPC, N-(3-nitrobenzyloxy259

0003-2697/82/040259-03$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserve&

260

CHRISTOPHE, TABLE

COMPARISON PAPERS

(B)

(A)

ACCORDING

AS DESCRIBED

TO ALWINE

Amount

Bound to Recovered Rc~~ercd washes

papr after washes in wskr washes in formamide (+watcr rinsings)

OF DBM

IN THIS

PAPER’

of DNA

(&g)

3.35 (49.3)b 3.17 (46.6)

6.16 (90.6) 0.52 (7.6)

0.24 (3.5)

0.11 (1.6)

AND

’ Amount of DNA present in each 6xaxation: 6.8 w (100%) bPerccntage8 given in parenthex%

RESULTS

TABLE CAPACITY

OF DBM

PAPER

2 WITH

INCREASING

Amount Amount of DNA incubated with 1 cm* of DBM paper: Bound to paper (after washes): Recovered in water washes: Recovered in formamide washes (+water rinsings): a Percentages

given

in parentheses.

11.6

(100)

10.85 (93.5) 0.3 (2.7) 0.4

(3.4)

AND DISCUSSION

The procedure described earlier by Alwine et al. (1,2) for the preparation of DBM paper has been modified in the following way in order to optimize the yield of each step. First, water was replaced by N,N-dimethylformamide (DMF) an aprotic solvent, as the medium for the nucleophilic displacement of the m-nitrobenzyloxymethyl group in order to eliminate a competing hydrolysis of the pyridinium salt (the immediate release of pyridine, easily identifiable by its odor when NBPC is dissolved in water, is indicative of this reaction). After heating, the remaining organic materials were removed by two washes with acetone. Second, following two washes with water, the reduction of the aromatic nitro function by sodium dithionite was carried out in 50 mM sodium hydroxide at room temperature rather than in water at 60°C. The use of an alkaline

(1 ml/cm’/wash) with a cooled solution of 1 M sodium acetate, pH 4, at O”C, blotted dry and soaked (20 pi/cm’) with a solution of denatured DNA in 1 M sodium acetate, pH 4, in a siliconized-glass vial. The vial is stoppered and incubated overnight at 4”C, allowing fixation to take place. Afterwards, the paper is washed three times with water (1 ml/cm2/wash) at room temperature, then with deionized formamide (500 pl/cm2/ wash), twice at room temperature and once at 68°C for 2 min, and finally rinsed three times with water (1 ml/cm2/wash) at room temperature. To test the binding capacity of the paper, plasmid DNA was radioactively labeled by nick translation to a specific radioactivity of 8.104 cpm/pg and monitored by ,&scintillation counting. The labeled DNA was de-

TEST OF THE BINDING

VASSART

natured by heating for 15 min at 70°C in 0.33 M sodium hydroxide, followed by the addition of 1 M Hepes (pH 7.5) to a final concentration of 0.12 M and neutralization with hydrochloric acid at 0°C. After ethanol precipitation, the DNA was dissolved in 1 M sodium acetate, pH 4, and this solution was briefly heated to 100°C and immediately cooled on ice before addition to the DBM paper.

1

OF THE PERFORMANCES

PREPARED

ET AL. AND

BROCAS,

of DNA

AMOUNTS

OF DNA

(pg)

22.5

(100)

44

(100)

21.15 0.4

(94) (1.7)

39.95 0.5

(90.8) (1.15)

0.9

(4.1)

3.45 (7.85)

IMPROVED

SYNTHESIS

medium prevents any acidification of the reaction mixture which would lead to the destruction of the reducing agent. Unnecessary heating is also avoided. After four washes with water and two washes with acetone to facilitate the subsequent drying, the ABM paper was stored at -20°C until final activation just prior to use. Diazotization was carried out in 1 M hydrochloric acid at 0°C using a stoichiometric equivalent of nitrous acid as calculated from the amount of NBPC engaged in the first step. After a short wash with 1 M sodium acetate, pH 4, and partial drying, the DBM paper was immediately incubated with denatured DNA at 4°C overnight. Free unreacted DNA was removed by three washes with water at room temperature. The paper turned orange uniformly at this stage, suggesting that it had been uniformly activated. (The orange color results from the presence of azo derivatives, which may be formed by a coupling reaction between two aromatic residues, one bearing a diazonium function and the other a hydroxyl group generated by hydrolysis of the diazonium function.) Since some renaturation occurs during the fixation step, further washing in denaturing conditions is needed to remove the unreacted DNA retained on the paper by hybridization. This was accomplished by three washes with formamide, two at room temperature and one at 68°C followed by :hree washes with water. When radioactively labeled DNA (6.8 pg) was incorporated in the fixation reaction and monitored in subsequent steps by counting, 90.6% of the radioactivity remained bound to the 0.5cm2 paper after washing (the three water washes removing most [7.6%] of the unbound [9.4%]), and the last wash only contained 0.03% of the input radioactivity. To compare the performance of this DBM paper to that of the DBM paper prepared according to Alwine et al. (1,2), 0.5 cm* of each type of paper was incubated with the same amount of radioactive DNA. The two paper fragments were then processed as de-

261

OF DBM PAPER

scribed above and the effectiveness of the binding measured by counting. The results summarized in Table 1 demonstrate clearly that the modified method leads to a DBM paper with increased binding capacity. Although we could never reproduce it, Alwine et al. (1,2) reported a binding capacity of 16-24 fig/cm2 for the DBM paper they prepared. In order to test the ability of the DBM paper prepared by ourselves to bind large quantities of DNA, three l-cm2 pieces of with increasing paper were incubated amounts of denatured DNA. The results shown in Table 2 again demonstrate the binding capacity of this DBM paper was higher than that reported previously. Finally, DBM paper prepared according to the method described here has been used successfully to characterize recombinant plasmids containing inserts of rat (4) and human (5) thyroglobulin cDNA by the hybridization-elution-translation technique, and to detect rat thyroglobulin mRNA sequences by the Northern Blotting method. ACKNOWLEDGMENTS The continuous support and interest of Dr. J. E. Dumont is greatly acknowledged. This work was supported by a contract Minis&e de la Politique ScientifiqueU.L.B. to Dr. J. E. Dumont, by NIH Grant AM 21732 and FRSM Grant 3.4334 to G.V.

REFERENCES 1. Alwine, J. C., Kamp, D. J., and Stark, G. R. (1977) Proc.

Nat.

Acad.

Sci. USA

14, 5350-5354.

2. Alwine, J. C., Kamp, D. J., Parker, B. A., Reiser, J., Renart, J., Stark, G. R., and Wahl, G. M. (1980) in Methods in Enzymology (Wu, R., ed.), Vol. 68, pp. 220-242, Academic Press, New York. 3. Thomas, P. S. (1980) Proc. Nat. Acad. Sci. USA 77, 5201-5205. 4. Brocas, H., Christophe, D., Van Heuverswijn, B., Scherberg, N., and Vassart, G. (1980) Biochem. Biophys. Res. Commun. 96, 1785-1792. 5. Brocas, H., Rocmans, P., Christophe, D., Van Heuverswijn, B., Pohl, V., and Vassart, G., manuscript in preparation.