Quantitative molecular hybridization on nylon membranes

ANALYTICAL BIOCHEMISTRY 149, 229-237 (1985)

Quantitative Molecular Hybridization on Nylon Membranes

GORDON CANNON, SABINE HEINHORST, AND ARTHUR WEISSBACH

Department of Cell Biology, Roche Institute of Molecular Biology, Roche Research Center, Nucley, New Jersey 071 IO

Received December 26, 1984

A study of DNA hybridization to DNA covalently bound to nylon membranes was made in order to develop a quantitative method for molecular hybridization using a nylon-based matrix. Chloroplast DNA was covalently attached to nylon membranes by irradiation at 254 nm. Under hybridization conditions the initial rate of DNA loss from the nylon membranes was 5-10s per 24 h, while under comparable conditions DNA bound to nitrocellulose membranes was lost at a rate of 38 to 6 1% per 24 h. Several sets of hybridization conditions were examined to select one giving reasonable hybridization rates and minimal loss of bound DNA. Under the conditions selected [Denhardt’s solution (D. Denhardt, 1966, Biochem. Biophys. Res. Commun. 23, 641-646) 0.5 M NaCI, 0.1% sodium dodecyl sulfate, and 31.4% formamide at 50°C for 92 h], hybridization was observed to be 29% more efficient on nylon membranes ihan on nitrocellulose. Several attempts to remove previously hybridized DNA from nylon membranes proved only partially successful. Reuse of the membranes, therefore, was of limited value. Quantitative hybridization of total radiolabeled tobacco cellular DNA to cloned tobacco chloroplast DNA attached to nylon yielded results similar to those previously reported using nitrocellulose membranes. However, use of nylon membranes greatly facilitated the manipulations required in the procedure. o 1985 Academic press, IIIC.

KEY WORDS: quantitative hybridization; chloroplast DNA; nylon membrane.

Molecular hybridization of nucleic acids to nitrocellulose membrane-bound DNA is perhaps the most common means of quali- tatively assessing the presence of a particular polynucleotide sequence in a mixture of nucleic acids (1). Usually, DNA is bound to nitrocellulose by passing a salt solution containing the denatured DNA through a nitrocellulose membrane. The denatured DNA is retained by the membrane and the binding procedure is completed by drying the membrane (2,3). Although DNA so prepared is sufficiently bound for qualitative hybridizations, the mechanism of binding is unknown and presumed to be noncovalent (1). The major disadvantages of nitrocellulose (i.e., fragility after drying and the noncovalent nature of the bond between it and DNA) have led to the development of alternate solid hybridization support matrices (4-6). In particular, nylon or chemically modified nylon

has been offered by a number of manufac- turers.’ DNA can be covalently bound to nylon by reaction of primary amino groups on the nylon with thymidine residues in the presence of 254-nm light (7). Recently Church and Gilbert (8) have detailed the transfer of DNA from acrylamide gels to nylon, its covalent attachment by ultraviolet irradiation and subsequent hybridization.

We have previously developed a method of quantitative molecular hybridization that has allowed us to measure the levels of chloroplast DNA in light-grown and dark- grown plant cells (results to be published elsewhere) and to study the effects of various metabolic inhibitors on chloroplast DNA synthesis in tobacco suspension cultures (9).

’ i.e.. Gene Screen and Gene Screen Plus from New England Nuclear, Boston, Mass., and Zeta-Probe from Bio-Rad Laboratories, Richmond, Calif.

229 0003-2697/W $3.00 Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

230 CANNON, HEINHORST, AND WEISSBACH

In order to develop a more facile hybridization procedure using the new nylon matrices, we carried out a study of the conditions required for DNA-DNA hybridization with cloned homologous DNA bound to nylon membranes. With this method we have quantitatively determined the chloroplast DNA content in cultured tobacco cells.

MATERIALS AND METHODS

Materials. All radiochemicals were pur- chased from Amersham Corporation (Arling- ton Heights, Ill.). Formamide was a product of Fluka Chemical Corporation (Hauppauge, N. Y.). Ficoll (average M, 400,000) was from Pharmacia (Piscataway, N. J.). Polyvinylpyr- rolidone (PVP)’ was from Sigma Chemical Company (St. Louis, MO.). Bovine serum albumin (BSA) was from Miles Laboratories (Elkhart, Ind.). Gene Screen Plus and Aquasol were obtained from New England Nuclear Corporation (Boston, Mass.).

Preparation of DNA. Radiolabeled DNA used in binding experiments to nitrocellulose or nylon membranes was extracted from Nicotiana tabacum suspension cells grown in the presence of [3H]thymidine. The DNA was isolated by a modification of the Murray and Thompson procedure previously described (9). DNA obtained from this procedure is larger than 27.3 kb as determined by agarose gel electrophoresis.

The radiolabeled tobacco DNA samples which were hybridized to membranes containing DNA were prepared according to the procedure of Dellaporta et al. (10). The final nucleic acid precipitate was rid of RNA and sheared to an average length of 500 nucleo- tides by treatment at 100°C for 3 min in 0.3 N NaOH. Recombinant plasmids containing PstI fragments of N. tabacum chloroplast DNA (2A-20.2 kb, 3A-19.2 kb, 4A-9.3 kb, 5A-8.2 kb, and 6A-6.9 kb) were isolated as previously reported (9). Bacterial clones con-

* Abbreviations used: SDS, sodium dodecyl sulfate; PVP, polyvinylpyrrolidone; SSC, sodium citrate-sodium chloride buffer; BSA, bovine serum albumin.

taining the recombinant plasmids with the above chloroplast DNA fragments were obtained from R. Fluhr (11).

Preparation of membranefilters containing DNA. DNA was adsorbed onto nitrocellulose membranes (Sartorius type SM P12; 2.5 cm; 0.45~pm pore size) and baked at 80°C as previously reported (9). Nylon membrane disks (2.2 cm) were cut from sheets of Gene Screen Plus. The recombinant plasmids containing tobacco chloroplast DNA fragments were mixed in equimolar amounts in 1.5 ml of 0.3 N NaOH and placed in a 100°C water bath for 3 min. After it was cooled in ice water, the solution was neutralized to pH 7.5 with 0.3 N HCl and 50 mM Tris-HCl (pH 8.0). The size of this denatured DNA ranged from 1.5 to 10 kb as determined by agarose gel electrophoresis. This denatured DNA (equal to 5 pg of chloroplast DNA fragments) was applied to the “B” side of the membrane (see instructions supplied with Gene Screen Plus) and dried under a heat lamp. The membranes were further irradiated with 254- nm light (flux of 320-400 pW/cm*) for periods of 1 to 25 min as indicated in the text, using a Model TS- 15 shortwave transillumi- nator (Ultraviolet Products, San Gabriel, Calif.). Ultraviolet flux was measured by a Blak-Ray J-225 shortwave uv meter (Ultra- violet Products). Immediately after the ultraviolet irradiation, unbound DNA was removed from the membranes by washing the filters in low salt buffer [ 10 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS)] at 65 “C for 15 min. The membranes were blotted, dried under a heat lamp, and stored desiccated in the dark.

Hybridizations. Hybridizations were conducted under one of two sets of conditions. In the first, the prehybridization solution consisted of 3X sodium citrate-sodium chloride buffer [SSC (1 X SSC contains 0.15 M

NaCl and 0.015 M Na citrate)], Denhardt’s solution [0.2% Ficoll, 0.2% PVP, 0.2% BSA (12)], 0.1% SDS, and 2 tI’tM EDTA at pH 7.2. Formamide was included in the solution at the concentrations listed in the text. Mem-

DNA HYBRIDIZATION ON NYLON MEMBRANES 231

branes used in this hybridization regime were prehybridized in sealed 20-ml glass scintillation vials containing the above solution for 4 h at the same temperature as that indicated for hybridization. The prehybridization solution was removed by aspiration and re- placed with hybridization solution (prehybridization solution containing 50 &ml denatured calf thymus DNA). Approximately 0.7 pg of 3H-labeled total cellular tobacco DNA was added and the membranes were incubated at the required temperatures and times. The membranes were washed twice at room temperature in 1X SSC (pH 7.6) containing 0.1% SDS and twice at 37°C in 0.2X SSC, 0.1% SDS. The membranes were blotted and dried, and the amount of radiolabeled DNA hybridized was determined by liquid scintillation counting in Beckman Redi-Solv NA. The second hybridization regime was conducted in 0.5 M NaHP04 (pH 7.2), 2 IItM EDTA, and either 1 or 7% SDS. The membranes were prehybridized in scintillation vials containing 2 ml of this hybridization solution for 15 min at 65°C. Hybridization was initiated by removing the prehybridization fluid and replacing it with 1 ml of fresh hybridization solution containing 0.7 pg of 3H-labeled total cellular tobacco DNA and placing the vial at 65°C for the indicated period of time. The membranes were washed, dried, and counted as described for the first protocol. In both protocols a blank filter containing no DNA was included in each vial to measure nonspecific binding of [3H]- labeled DNA to the membranes.

Stability of membrane-bound DNA. Mem- branes containing 5 pg of 3H-labeled DNA attached by ultraviolet irradiation (nylon membranes) or baking (nitrocellulose membranes) were exposed to hybridization conditions as detailed above, and the amount of DNA remaining bound to the membrane after various incubation times was determined by liquid scintillation counting.

Removal of hybridized DNA from nylon membranes. Hybridized ‘H-labeled DNA was eluted from nylon-bound DNA by washing

the membranes for various times in the solutions listed in Table 3. The membranes were washed with agitation twice in volumes that exceeded 10 ml per membrane. The percentage of hybridized DNA remaining bound after the elution was determined by drying the membrane and counting its radioactivity on the membrane.

RESULTS

Binding of DNA to Nylon Membranes

The binding of DNA to nylon membranes was assessed by loading 5 pg of 3H-labeled denatured tobacco DNA onto nylon membrane disks and irradiating with ultraviolet light for increasing times up to 25 min (Fig. 1). The disks were subsequently washed in 10 mM Tris-HCl (pH 8.0) containing 2 mM EDTA and 0.1% SDS at 65°C for 15 min. The amount of labeled DNA remaining was determined as described under Materials and Methods. In the absence of ultraviolet irradiation 44% of the DNA attached to the membrane was removed when washed at 65°C (Fig. 1). Irradiation increased the percentage of DNA resistant to the elution at 65°C to 80% of the amount loaded. Irradia- tion times greater than 10 min (1.8 kJ/m’)

‘OOv

1, I I, II 1, I, II 0 4 6 12 16 20 24

UV IRRADIATION hid

FIG. 1. Binding of single-stranded DNA to nylon membranes with uv irradiation. Single-stranded ‘H-labeled DNA (5 r.& was dried onto 2.2-cm nylon membrane disks and exposed for increasing times to uv irradiation (254 nm, 360 pW/cm*). The filters were washed at 65°C for 15 min in buffer containing 10 mM Tris-HCI, pH 8.0, 2 mM EDTA, and 0.1% SDS. After drying, the DNA remaining bound to the membranes was measured by liquid scintillation counting.

232 CANNON, HEINHORST. AND WEISSBACH

did not result in significant increases in bound DNA (78.5% bound at 10 min to 80.2% bound after 25 min). Therefore, 7.5 min (1.4 kJ/m’) was chosen as the standard irradiation time used to bind DNA to the nylon membranes in these studies.

Stability of Nylon Bound DNA to Hybridization Conditions

Nylon and nitrocellulose membrane disks containing 5pg of ‘H-labeled tobacco DNA were subjected to a variety of hybridization conditions to measure the stability of the membrane bound DNA. Two different sets

of conditions were tested. The first tests (numbers 1-6, Table 1) were conducted in hybridization solutions containing Denhardt’s solution (12), 0.5 M NaCl, 0.1% SDS, and formamide. Formamide concentrations and the reaction temperature were varied as indicated in Table 1. The second group of reactions (numbers 7 and 8, Table 1) were conducted in 0.5 M NaHP04 (pH 7.2) at 65 “C in the presence of either 7 or 1% SDS. After treating the membranes for periods of 24-64 h under the listed conditions, the disks were removed and washed and the amount of labeled DNA remaining on the disks was determined as described under Materials and

TABLE 1

Loss OFDNA FROMMEMBRANESDUR~NGTREATMENTTOHYBRIDIZAT~ONCOND~TIONS

Number Conditions

Cycle 1

Length of treatment 76 of loaded

00 DNA lost

Cycle 2

Length of 90 of loaded treatment DNA lost

(h) (Cycles I + 2)

Cycle 3

Length of 9’0 of loaded treatment DNA lost

(h) (Cycles 1 + 2 + 3)

Nylon membrane, 50% formamide, 31°C

Nylon membrane, 45.7% formamide, 40°C

Nylon membrane, 38.6% formamide, 45°C

Nylon membrane, 3 1.4% formamide, 50°C

Nitrocellulose membrane, 50% formamide, 37°C

Nitrocellulose membrane, 0% formamide. 65°C

Nylon membrane 7% SDS, 65°C

Nylon membrane 1% SDS, 65°C

69 14.4 42 17.4 92 30.2

69 19.0 42 23.4 92 30.9

69 18.2 42 26.3 92 35. I

69 27.0 42 31.2 92 33.9

24 38.1 48

24 60.7 48

48

64

48.1

25.7

48

84

43.1 - -

73.5 - -

55.1 - -

36.1 - -

Note. Radiolabeled DNA was bound to membrane filters as described under Materials and Methods and exposed to the indicated hybridization conditions, After the washing and drying, the percentage of DNA remaining bound to the membrane was determined as described under Materials and Methods.


Methods. The disks were then used for a second or third cycle of treatment. Table 1 contains the actual observed loss of DNA from the filters during two and three cycles of mock hybridization. From the data in Table 1, it is apparent that nylon membranes incubated at 37-50°C in aqueous formamide mixtures lost 14-27% of their DNA (experiments 1-4, Table l), whereas those incubated at 65” lost 25-48% (experiments 7 and 8, Table 1) during the first cycle of hybridization. Furthermore, a high SDS concentration (7%) increases the loss of DNA from the nylon membrane when compared to incubations in 1% SDS. Moreover, the DNA loss from the membranes decreased significantly after the initial cycle of treatment, suggesting that some noncovalently bound DNA was not removed by the original 65°C wash in 12 mM salt which was carried out immediately after binding of the DNA to the membrane. Nylon membranes treated in solutions containing Denhardt’s solution and formamide, at temperatures of 37-50°C retained 65- 70% of their DNA after 3 cycles of hybridization conditions for a total time of 203 h. In contrast, nitrocellulose membranes were observed to lose 38-60% of their DNA after 24 h of treatment (experiments 5 and 6, Table 1). As with the nylon membranes after the initial cycle under hybridization conditions, the DNA loss from nitrocellulose was reduced in subsequent cycles of treatment.

Rates of Hybridization to DNA Bound on Nylon Membranes

Cloned tobacco chloroplast DNA fragments were covalently bound to nylon membranes by ultraviolet light. The membranes were then hybridized to 3H-labeled total cellular DNA isolated from tobacco suspension cells. Hybridizations were conducted in the presence of Denhardt’s solution (12), 0.5 M NaCl, 0.1% SDS at the indicated formamide concentrations and temperatures (Fig. 2). Fil- ters were removed at various times up to 116 h and the percentage of input radiolabeled

FIG. 2. Comparison of hybridization rates on nylon membranes at different temperatures. Hybridizations were conducted at the indicated temperatures in Den- hardt’s solution (12) containing 0.5 M NaCl and 0.1% SDS. Formamide concentrations were varied in order to maintain hybridization conditions equivalent to 72°C in 100% aqueous solution and 0.5 M NaCl. After the hybridization, the membranes were washed twice for 15 min in 1 X SSD, 0.1% SDS at room temperature and twice for 15 min in 0.2 X SSC, 0.1% SDS at 37°C. The membranes were then dried under a heat lamp and the hybridized ‘H-labeled DNA was determined by liquid scintillation counting. (A) 31.4% formamide, 50°C; (A) 38.6% formamide, 45°C; (0) 45.7% formamide, 40°C; (0) 50% formamide, 37°C.

DNA hybridized was measured. The temperature required for duplex formation was ad- justed by varying the concentration of formamide so that the hybridizations were all conducted at the equivalent of 72°C in 0.5 M NaCl (Fig. 2).

Despite the compensating adjustments in formamide concentrations, the temperature was found to markedly influence the hybridization rates, i.e., higher temperatures resulted in increased hybridization rates (Fig. 2).

Comparison of Hybridization on Nitrocellulose and Nylon Membranes

Hybridization rates of 3H-labeled total cellular tobacco DNA to cloned tobacco chloroplast DNA attached to either nitrocellulose or nylon membranes were determined under two sets of conditions for nylon membranes


[l% SDS, 0.5 M NaHPO.,, pH 7.2, 2 IIIM EDTA at 65°C or Denhardt’s solution (12), 0.5 M NaCl, 31.4% formamide, and 0.1% SDS at 5O”C] and under the conditions previously reported for nitrocellulose (see Materials and Methods). Hybridization of 3H-labeled DNA to nitrocellulose-bound DNA was observed to reach a maximum of 2.8% of input DNA after 63 h, but with increasing incubation time the value dropped to a value of 2.5% after 116 h (Fig. 3). Hybridization to nylon-bound DNA in 1% SDS at 65°C reached a maximum of 4.5% after 92 h and dropped to 4.1% after 120 h. Hybridization with nylon membranes at 50°C in solutions containing 3 1.4% formamide and Denhardt’s solution reached a maximum of 3.6% after 92 h and this level of hybridization was maintained through 116 h. In hybridizations to nylon-bound DNA under both experimental conditions, the maximum level of hybridization was 28-61% higher than that for nitrocellulose.

An internal control probe consisting of 0.1 ng 32P-labeled DNA identical to that immobilized on the nylon membrane was included in a hybridization mixture to determine the efficiency of hybridization in Denhardt’s solution, 31.4% formamide, 0.5 M NaCl, 0.1%

* oou HOURS

FIG. 3. Comparison of hybridization rates on nitrocellulose membranes in Denhardt’s solution (12), 50% formamide, 0.1% SDS, 0.5 M NaCl at 37°C (0 - 0) and on nylon membranes in either 1% SDS, 2 mM EDTA, 0.5 M NaHP04, pH 7.2, at 65°C (0 - - - 0) or in 1 X Denhardt’s solution ( 12), 3 I .4% formamide, 0.1% SDS, 0.5 M NaCl at 50°C (0 - 0).

SDS at 50°C. The reaction was conducted for 62 h and the hybridization efficiency was found to be 53.4% (data not shown). Thus, the percentage of 3H-labeled total cellular DNA hybridized (2.3%) was corrected to account for the less than 100% efficiency of the hybridization and for the 46% of the chloroplast genome bound to the membrane (9). The percentage of total cellular DNA calculated to be chloroplast DNA was 8.1% which is similar to our previous measure- ments using nitrocellulose membranes (manuscript submitted).

Removal of Hybridized DNA from Nylon- Bound DNA

Nylon membranes containing 3H-labeled, hybridized DNA were washed in 10 mM Tris-HCl (pH 8.0) containing 2 mM EDTA and 0.1% SDS at 75 or 95°C as described under Materials and Methods. Only 76-79% of the hybridized DNA was removed under these conditions (Table 2). Moreover, attempts to remove greater proportions of the DNA by treatment of the membranes with alkali were even less successful since only 30% was removed by heating the membranes to 40°C in 0.4 M NaOH for 30 min. The inability to totally remove hybridized DNA from the membranes may have resulted from the drying step required for liquid scintillation counting of the membrane. The instructions included with Gene Screen Plus warn that total drying of the membrane may result in some degree of irreversible binding by hybridized DNA. An attempt to circumvent the drying steps was made in which the membranes were never allowed to dry and were counted as wet membranes in a scintillation fluid for aqueous samples (Aquasol). However, even in this case, it was only possible to remove about 25% of the hybridized radiolabeled DNA from the membranes by washing in 10 mM Tris-HCl (pH 8.0) containing 2 mM EDTA and 0.1% SDS at 95°C.


TABLE 2

REMOVAL OF HYBRIDIZED 3H-L~~~~~~ TOBACCO DNA FROM NYLON MEMBRANES

Elution conditons % of hybridized DNA

remaining after elution

15 min 75°C. 10 mM Tris, 0.1% SDS 21.1

20 min 95”C, 10 mM Tris. 0.1% SDS 25.1

15 min 25°C 0.1 M NaOH 98.0 30 min 42”C, 0.4 M NaOH 70.9

Note. Membranes with known quantities of radiolabeled hybridized DNA were exposed to the indicated treatment and after drying they were recounted to measure the amount of hybridized DNA remaining (as described under Materials and Methods).

Reuse of Nylon Membranes

Nylon membranes containing hybridized DNA were washed in 10 KIM Tris-HCl (pH 8.0) containing 2 mM EDTA and 0.1% SDS for 20 min at 75°C. The residual radioactivity on the membranes was in the range of 20- 80 cpm per membrane. The precise number of cpm on each membrane was recorded and later used to correct observed hybridization. The used membranes, as well as new control membranes, were used for hybridization at 50°C in hybridization solution containing Denhardt’s solution, 3 1.4% formamide, 0.1% SDS, and 0.5 M NaCl. The membranes were removed from the hybridization at various times from 60-l 34 h (Fig. 4). As is seen in Fig. 4, hybridization proceeded to a maximum of 3.2% of the input [3H]DNA and no significant difference in rate was observed between previously used and new membranes.

DISCUSSION

The results reported here assess the use of nylon membranes as a solid support matrix for quantitative mixed-phase hybridizations. It was observed that elevated hybridization temperatures (65°C) in conjunction with high SDS concentrations (7%) accelerate the loss

of DNA immobilized on nylon membranes. Reduction of the SDS concentration to 1% resulted in a lowered rate of DNA loss. The removal of the DNA bound to nylon was further reduced by conducting hybridizations at 37-50°C in solutions containing 0.1% SDS and Denhardt’s solution and formamide. However, this also reduced the rate and maximum level of hybridization (Fig. 3). The presence of SDS in the hybridization solution reduces nonspecific binding by radiolabeled DNA (8). Lower (0.1% compared to 1 or 7%) SDS concentrations resulted in no significant increase in background counts under the conditions employed here; therefore, it was concluded that SDS concentrations above 0.1% were unnecessary to maintain low background counts. If one uses 0. I- 1 .O% SDS in both low (37-50”(Z) and high (65°C) temperature hybridizations, nitrocellulose membranes are found to lose bound DNA to a greater extent (38-61%) than nylon membranes (14-27%) during the first cycle of hybridization. This reflects the probable noncovalent nature of the bond between DNA and nitrocellulose. Both nitrocellulose and nylon membranes lost a larger proportion of bound DNA during the first hybridization

HOURS

FIG. 4. Comparison of hybridization rates on once- used nylon membranes (0 - 0) and new ones (0 - - - 0). Hybridized [‘H]DNA was partially eluted from used membranes at 75°C as described under Ma- terials and Methods. Residual radioactivity was determined for each used membrane and subtracted from the observed hybridized counts in this hybridization. Hy- bridizations were conducted at 50°C in Denhardt’s solution (12), 0.1% SDS and 0.5 M NaCl as described under Materials and Methods.


cycle than in subsequent cycles. Since the loss of DNA from nylon membranes in the later cycles was significantly lower, it must be concluded that the DNA lost during the first cycle represented incompletely bound DNA that was not removed by the 65°C wash conducted immediately following the binding procedure.

Rates of DNA hybridization on nylon membranes in the 37-50°C range were noted to increase with increasing temperature. Al- though formamide was added to the hybridization solutions at concentrations designed to lower the temperature required for duplex formation (13) and therefore compensate for the different temperature of the reaction, the rate of hybridization remained dependent upon temperature. Likewise, enhanced rates and maximal levels of hybridization were observed at 65°C in 1% SDS and 0.5 M NaHP04. These facts suggest to us that a steric interaction hindering hybridization takes place between the nylon and the membrane-bound DNA. This steric hindrance is reduced at higher temperature and allows hybridization to take place at a greater ve- locity.

Comparison of hybridizations of nitrocellulose membranes (at 37°C in 50% formamide, Denhardt’s solution, and 0.5 M NaCl) with those conducted on nylon (at 65°C in 1% SDS or at 50°C in 31.4% formamide, Denhardt’s solution, and 0.5 M NaCl) proved that hybridization on nylon membranes results in higher maximum percentages of input DNA hybridized (Fig. 3). This is similar to the increased levels of hybridization observed with DNA covalently bound to diazobenzyl- oxymethyl paper ( 14). However, it was also noted in both the 65°C nylon reaction and the nitrocellulose reaction that hybridization proceeded to a maximum level and then decreased with further incubation, The decreased levels was possibly due to membrane- bound DNA being lost to the liquid phase. Thus, the selection of optimal hybridization conditions is complicated by the observation

that the rate and level of hybridization is enhanced at higher temperatures, but this also increases the loss of membrane-bound DNA. We found the optimal compromise conditions were at a temperature of 50°C in Denhardt’s solution containing 0.1% SDS, 3 1.4% formamide, and 0.5 M NaCl. By using these conditions, we were able to determine the percentage of chloroplast DNA in cellular DNA isolated from tobacco suspension cultures to be 8.1%. This value agrees well with that (8.3%) which we had previously obtained using nitrocellulose membranes (manuscript submitted).

One supposed feature of nylon that should enhance its utility as a hybridization support matrix is the reported ability to remove previously hybridized radiolabeled DNA to allow the membrane-bound DNA to be used again (8). Our results indicate that 70-80% of the hybridized DNA can be eluted by washing the membranes in low salt (12 mM salt) at 75°C. Since the quantitative procedure used in our investigations uses liquid scintillation counting to measure the amount of hybridized DNA, the residual radioactivity remaining from a previous hybridization complicates interpretation of the observed data when the membranes are reused. We, therefore, con- clude that reuse of the membranes for quantitative hybridizations is impractical, except for special cases in which the initial hybridizations involve low radioactivity and residual counts per minute are therefore low. How- ever, it should be noted that when we reused membranes that had previously been employed in hybridization reactions, we were able to observe no differences (Fig. 4) in the amount or rate of hybridizations between previously used membranes and those that were new.

Nylon membranes are quite resistant to the mechanical manipulations required for quantitative hybridizations and are in marked contrast to nitrocellulose membranes which become brittle and are easily broken after drying. Furthermore, the DNA-binding pro-


cedure required for nylon membranes was 5. Saris, C., Franssen, H., Heuyerjans, J., Eenbergen,

significantly less time consuming than that J., and Bloemers, H. (I 982) Nucl. Acids Rex 10,

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istics, taken with the enhanced performance 6. Seed, B. (1982) Nucl. Acids Rex 10, 1799-1810. 7. Saito, I.. Sugiyama, H., Furukawa, N., and Matsaura,

of hybridizations conducted on nylon, dem- T. (198li Tetrahedron Lett. 22, 3265-3268.

onstrate that nvlon is a sunerior alternative 8. Church, G. M., and Gilbert, W. (1984) Proc. Nutl.

to nitrocellulose as a solid support matrix for Acad. Sci. USA 81, 1991-1995.

quantitative molecular hybridization. 9. Heinhorst, S., Cannon, G. C., and Weissbach, A. (1985) Plant Mol. Biol. 4, 3-12.

10. Dellaporta, S. L., Wood, J., and Hicks, J. B. (1983)

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Quantitative molecular hybridization on nylon membranes

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