Binding of DNA to cellulose nitrate filters under denaturing conditions

ANALYTICAL

BIOCHEMISTRY

Binding

78, 569-571 (1977)

of DNA to Cellulose Nitrate Filters under Denaturing Conditions

Conditions are described for the immobilization nitrate filters.

of DNA in alkali to cellulose

It appears useful to bind DNA to cellulose nitrate filters under denaturing conditions. Under the conditions described here, bimolecular reassociation of rapidly reannealing DNA sequences and unimolecular foldback of inverted-repeat sequences should be reduced or prevented in the time period after denaturation and prior to fixing of the DNA on filters. METHODS

In the standard procedure used, Millipore HAWP, 0.45 pm, 24-mm filters were first floated on and then soaked in distilled water for at least 15 min prior to use, DNA, denatured in 0.2 M NaOH for 5 min at room temperature, was adjusted to 2.0 M NaCl, 0.2 M NaOH. The soaked filters were washed under vacuum filtration with 15 ml of water followed with 5 ml of 2.0 M NaCl. Immediately, with the filter vacuum off, a volume of solution required to load a desired amount of DNA was layered onto a filter. Under low vacuum allowing a flow rate of about 1 ml/min, the DNA solution was passed through the filter. With normal water aspirator vacuum, filters were then washed with 15 ml of 2 M NaCl. The filters were dried at 80°C under vacuum. RESULTS

Figure 1 shows the percentage of 14C-labeled E. co/i DNA retained on filters at various concentrations of NaCl in the 0.2 M NaOH loading solution. Maximum retention (about 85%) of this DNA occurred when the NaCl concentration was above 1.5 M. For different quantities of 14C-labeled E. coli DNA immobilized by the standard procedure described in Methods, Table 1 shows the percentage of DNA retained on filters after a 16-hr period at 67°C in 6x SSC. (SSC, standard saline citrate, is 0.15 M NaCl, 0.015 M sodium citrate.) Approximately 82% of the DNA immobilized on filters at high pH was retained after this incubation period for any of the quantities of DNA tested. This compared to about 87% retained when the DNA had been mounted on filters at neutral pH by the method of Gillespie and Spiegelman ( 1) . 569 Copyright 0 1977 by Academic Press. Inc. All right5 of reproduction in any form reserved.

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FIG. 1. The effect of NaCl concentration on the binding of DNA to cellulose nitrate filters under alkaline conditions. ‘Y-Labeled E. co/i DNA, 25.2 pg, was applied to each filter as described in the standard procedure. The DNA solution was 0.2 M NaOH with various concentrations of NaCl as indicated on the abscissa of the graph. The filters were washed and dried as described in Methods and then counted in toluene scintillation fluid. The percentage of DNA retained on each filter was calculated by comparison of the amount of radioactivity on these filters with the radioactivity measured on Millipore filters bearing DNA precipitated with 10% trichloroacetic acid in the presence of bovine serum albumin carrier.

Figure 2 compares the hybridization ability of DNA immobilized by the standard procedure with the hybridization ability of DNA immobilized by the method of Gillespie and Spiegelman (1). 3H-Labeled E. coli RNA was hybridized to immobilized E. coli DNA as described under Fig. 2. These results show that the efficiency of hybridization of DNA immobilized by either of the two methods is essentially the same. TABLE Loss OF DNA FROM FILTERS Amount of DNA per filter before incubation (wg)

1 DIJRING~NCUBATION~

DNA retained on filters after incubation (%)

Method A

Method B

Method A

Method B

16.1 32.7 65.4 128.7

16.0 31.6 64.8 133.7

83 79 82 84

88 89 87 85

u Escherichia co/i [14C]DNA was immobilized on filters under denaturing conditions by the standard procedure described in Methods (method A in this table) or by the method of Gillespie and Spiegelman (1) (method B in this table). Filters were incubated in 6 x SSC for 16 hr at 67°C. washed three times in large volumes of 6x SSC, dried, and counted in toluene scintillation fluid. The percentage of DNA retained after incubation is based on the amount of DNA retained on a similar filter which had not been incubated or washed.

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FIG. 2. Hybridization of [3H]RNA to DNA immobilized on filters. 20.1 pg of alkali-denatured E. coli DNA was applied to each filter either under denaturing conditions (- 0 -) by the standard procedure described in Methods or at neutral pH (- l -) by the method of Gillespie and Spiegelman (1). E. co/i [3H]RNA was hybridized to the filters in 6x SSC at 67°C for the times shown. After hybridization, the filters were treated with heated pancreatic RNase (10 pg/ml in 2 x SSC) for 1 hr at room temperature, washed three times in large volumes of 6x SSC. dried, and counted. The 3H retained by blank filters has been subtracted.

DISCUSSION

It has previously been shown that alkali denaturation of DNA is not affected by high concentrations of DNA or salt (2,3). We show here that under conditions of high salt concentration, DNA in alkali can be efficiently immobilized onto cellulose nitrate filters. ACKNOWLEDGMENTS I thank Dr. Charles A. Thomas, Jr. for laboratory facilities and discussion.

REFERENCES 1. Gillespie, D., and Spiegelman, S. (1965) J. Mol. Biol. 12, 829-842. Ehrlich, P., and Doty, P. (1958) J. Amer. Chem. Sot. 80, 4251-4255. 3. Gillespie, D. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.) Vol. 12B, pp. 641-668, Academic Press, New York. 2.

ROBERT F. BAKER Department of Biological Sciences University of Southern California Los Angeles, California 90007 Received July 30, 1976; accepted

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1976