Biochimica et Biophysica Acta, 740 (1983) !-7 1 Elsevier

BBA91199

ASPECTS OF THE MECHANISM OF ACID-PHENOL EXTRACTION OF NUCLEIC ACIDS

DIETER MULLER, BERND HOFER, ANNIE KOCH and HUBERT KOSTER *

Institut fiir Organische Chemie und Biochemie, Universitiit Hambur~ Martin - Luther - King - Platz 6, D - 2000 Hamburg 13 (F. R. G.)

(Received October 29th, 1982)

Key words: DNA extraction; Acid-phenol extraction; Mg 2 + effect

Non-covalently dosed circular (ccc) DNA was found to be denatured at pH 4.0 in aqueous solutions saturated with phenol, while relaxed ccc DNA retained a completely double-stranded form. Under these conditions, increasing concentrations of magnesium ions are required to extract non-ccc, superhelical, and relaxed ccc DNA species from the aqueous into the phenolic phase. Extraction of RNA was not observed under any conditions. The contribution of phenol, protons, and metal ions to these effects are discussed.

Introduction

Recently a relatively simple method for the isolation of superhelical ccc DNA has been de- scribed [1], which is based on the use of phenol for selective extraction of non-ccc DNA at acidic pH. However, no explanation for this effect has been given. Using different types of DNA species (single-stranded; double-stranded linear; open cir- cular; superhelical closed circular; relaxed closed circular) we gained some insight into the mecha- nism underlying this phenomenon.

Materials and Methods

Chemicals. All chemicals were of p.a. grade. Phenol (Merck) was distilled under nitrogen. Agarose ('Seakem') was purchased from Marine Colloids.

Enzymes. HpalI endonuclease and S l nuclease were purchased from Miles.

Nucleic acids. Isolation of phage fd single- stranded DNA [2] as well as in vitro synthesis and

* To whom correspondence should be addressed. Abbreviation: ccc DNA, covalently dosed circular DNA.

0167-4781/83/$03.00 © 1983 Elsevier Science Publishers B.V.

purification of double-stranded fd DNA species (relaxed ccc, open circular, and finear) up to ethanol precipitation [3] have been previously de- scribed. Double-stranded fd DNAs were 3H- labeled in deoxyguanosine or deoxycytidine at a specific activity of 103-104 cpm per nmol of nucleotides. Superhelical fd DNA synthesized in vivo was a generous gift from J. Wrstemeyer of this laboratory. Fragmentation of double-stranded fd DNA with endonuclease HpaII was as de- scribed in Ref. 2. The fragments were purified in the same way as the double-stranded fd DNA. RNA species were purchased from Boehringer, Mannheim. All nucleic acids were dissolved in 1 mM Tris-HC1 (pH 8)/1 mM EDTA as proposed by Zasloff et al. [1].

Acid-phenol extraction. In the standard proce- dure DNA or RNA solutions (see above) were adjusted to 1.5 mM MgC12. 0.1 vol. of sodium acetate, (pH 4.0) (1 M in Na+), was added and the mixture was vortexed for a few s in 0.9 vols. of phenol, equilibrated with 10 mM Tris-HC1 (pH 7.5)/0.1 mM EDTA. After vortexing for 45 s the solution was centrifuged at 10000 × g for 1 min and the aqueous phase was withdrawn. (Devia- tions from this procedure are indicated in Results

and Discussion.) For preparative purposes the aqueous phase was immediately neutralized by the addition of 0.12 vols. of 2 M Tris base, and resid- ual phenol was extracted with ether. The organic phase was then withdrawn, and the reaction tube was treated with 10 mM EDTA (pH 7.5) for 3 rain at 80°C and vortexed for 5 min to dissolve poten- tial pelleted material.

Aqueous and organic phases as well as the 'pelleted solution' were analyzed by one or both of the following methods: (1) Liquid scintillation counting: Samples were applied to cellulosenitrate filters, dried, and counted in 5 ml of a toluene-based scintillation cocktail. (2) Agarose gel electrophoresis: Samples were elec- trophoresed in vertical or horizontal slab gels of 1% agarose in 40 mM Tris-HOAc/20 mM N a O A c / l mM EDTA/0 .5 / tg /ml ethidium bromide (pH 7.7) [4] at 80-100 V for 2-6 h. Visu- alization and photography of nucleic acid bands have been described earlier [3]. For quantification, the bands were cut out and the gel pieces were dissolved by incubating each with 500/~l of water at 95°C for 30 min. The samples were counted in 5 ml of a dioxane-based scintillation cocktail.

Polyacrylamide gel electrophoresis (4.5% acrylamide/7 M urea) was as described in Ref. 2.

S 1 nuclease reactions. DNA solutions were adjusted to 2 mM ZnSO 4 and saturated with phe- nol. 20 /~l samples, containing about 0.4 /~g of DNA, were mixed with 0.1 vols. of NaOAc (pH 4.0) (l M in Na ÷) and vortexed for 45 s. Then 1/~1 (10000 U) of enzyme was added. The mixture was incubated for 15 rain at room temperature. The reaction was stopped by addition of 1 vol. of dye-mix (10 M urea /20 mM EDTA/0.05% xylene-cyanol/0.05% Bromophenol blue) and the products were analyzed by agarose gel electro- phoresis (see above).

Results

Behaviour of DNA at p H 4.0 and 1.5 m M MgCI 2 To assay the behaviour of different DNA species

during acid-phenol extraction a mixture contain- ing relaxed ccc DNA, open circular DNA, and double-stranded linear DNA was routinely used. It was dissolved in a low ionic strength buffer

a b c d e f g

OC

ds l inear

re laxed c c c

ss l inear ss c i rcular

Fig. 1. Acid-phenol extraction assays with the complete and incomplete systems. 30 t.tl samples of 0.5 #g of double-stranded (ds) DNA species were subjected to the "acid-phenol extrac- tion' protocol described in Materials and Methods, but with the modifications given below. Aliquots of the aqueous phases were analyzed by agarose gel electrophoresis. Lanes a and g: refer- ences of double-stranded and single-stranded (ss) DNA species. respectively. Extraction procedure with the complete system (b), without Mg 2+ (c), without NaOAc, pH 4.0 (d), without phenol (e), without phenolic phase, but with a phenol-saturated aqueous phase (f). oc, open circular.

adjusted to 1.5 mM MgC12, and acidified by addi- tion of NaOAc (pH 4.0). Phenol was added, the mixture was vortexed and centrifuged. The aque- ous phase was withdrawn and analyzed by liquid scintillation counting and agarose gel electro- phoresis (for details see Materials and Methods). To gain some insight into the mechanism of extraction, single components of this system were systematically omitted. The results are summarized in Table I and Fig. 1. With the complete system all DNA species but relaxed ccc DNA were extracted from the aqueous phase (Table I, Expt. 2). When Mg 2+ was omitted, only a slight extraction of DNA was observed (see Table I), but drastic changes showed up in the electrophoretic pattern (Fig. 1, lane c). The bands of open circular and double-stranded linear DNA had vanished, while two new bands with the mobilities of single- stranded unit length linear and circular DNA had appeared. (As 90% of the radioactivity had re- mained in the aqueous phase, the discrepancy

TABLE I

ACID-PHENOL EXTRACTION ASSAYS WITH THE COMPLETE AND INCOMPLETE SYSTEMS

In Expt. 6 phenol was reduced to the amount necessary to saturate the aqueous solution, i.e. there was no phenolic phase, ss, single-stranded.

Expl. no.

Components of the system Components in aqueous phase after extrac- tion procedure

cpm DNA species see (%) Fig. 1

! DNA 100 ccc, non-ccc lane a 2 DNA/MgCI 2/NaOAc (pH 4)phenol 53 ccc lane b 3 DNA/2qaOAc (pH 4)/phenol 90 ccc, ss lane c 4 DNA/MgC12/phenol 95 ccc, non-ccc lane d 5 DNA/MgCI 2,/NaOAc (pH 4) 100 ccc, non-ccc lane e 6 DNA/MgCI2/NaOAc (pH 4)/phenol 86 ccc, ss lane f

between the intensities of the vanished and the new bands must be due to the fact that ethidium bromide staining of single-stranded D N A is much less efficient than of double-stranded DNA.) This finding indicates that selectively the non-ccc D N A species were converted into their single-standed forms ( 'denatured'). Moreover, it shows that the presence of Mg 2÷ at appropriate concentrations is essential for complete extraction.

To test whether denaturation occurred also in the presence of Mg 2÷, an experiment was per- formed in which the addition of phenol was limited to the amount necessary to saturate the aqueous solution (Table I, Expt. 6). Thus no organic phase could be formed, and D N A extraction should be impossible. In fact almost all of the D N A re- mained in solution. Non-ccc species were dena- tured just as in the absence of Mg 2÷.

When either the acidic buffer or the phenol were omitted neither denaturation nor extraction of D N A were observed (Table I, Expts. 4 and 5). Thus both, phenol and pH 4.0, are essential for the observed denaturation of non-ccc DNA. This means that phenol does not only provide an organic phase for the extraction, but, as it is partly soluble in water, also acts as a denaturing agent, possibly by disturbing base stacking.

It should be noted that the D N A extracted under our conditions was present neither in the interface nor in the organic phase, but as a pellet. Nonetheless the presence of a phenolic phase was

essential for extraction. Saturation of the aqueous solution with phenol without formation of an organic layer had no effect (see Table I, Expt. 6).

Behaviour of DNA at elevated MgCl 2 concentra- tions

Earlier experiments had demonstrated that MgCI: concentrations substantially greater than 1.5 mM lead to partial extraction even of relaxed ccc DNA. A detailed study of this phenomenon, using both relaxed and superhelical ccc DNA, is shown in Figs. 2 and 3.

Relaxed ccc D N A virtually remairied in the aqueous layer at MgC12 concentrations up to 20 mM (Fig. 2A). At 30 mM, however, the D N A was quantitatively extracted. This phenomenon was not due to nicking, because the D N A was resolved from the pellet (see Materials and Methods) in its coc form (Fig. 2B).

Superhelical D N A behaved quite differently. It was completely extracted already at 2 mM Mg 2+ (Fig. 3). Up to 1.5 mM Mg 2÷ most of this D N A remained in the aqueous phase (data not shown).

The Mg 2+ concentrations necessary for com- plete extraction of different D N A species are sum- marized in Table II.

The question, whether or not the extraction of relaxed ccc D N A occurs due to denaturation at elevated Mg 2+ concentrations [5], was examined using the single strand-specific endonuclease S I. Mixtures of relaxed ccc, open circular, and linear

4

TABLE II

MINIMAL Mg 2+ CONCENTRATIONS FOR COMPLETE EXTRACTION UNDER STANDARD CONDITIONS

DNA species Mg 2 + con-

centration (mM)

Non-ccc (fd) 1.0- 1.5 Superhelical-ccc ( fd) 1.5- 2.0 Relaxed-ccc ( fd ) 20 - 30

double-stranded D N A were incubated with nuclease S l under the conditions of acid-phenol extraction. To avoid extraction of the enzyme into the organic phase, addition of phenol was limited to the amount required to saturate the aqueous solution. The mixture was adjusted to 2 mM ZnSO 4 which is essential for S I activity.

Three experiments were performed with MgC12 concentrations of 0, l0 and 30 mM. Two of them are shown in Fig. 4, lanes c and e. In all cases the non-ccc DNAs were completely degraded. This

proves that the enzyme is active under these condi- tions and provides further evidence for the de- naturation of these DNA species. The relaxed ccc DNA, on the other hand, remained completely intact even at 30 mM MgCI2.

In control experiments without phenol it was observed that S 1 converts open circular DNA to double-stranded linear D N A by cutting one strand opposite to a nick or gap. But, as expected, no degradation of the double-stranded linear species takes place (Fig. 4, lanes b and d).

Behaviour of DNA at pH 7. 5 The finding that relaxed ccc DNA can be ex-

tracted without denaturation at 30 mM Mg 2÷ raised the question, if elevated MgCI 2 concentra- tions can lead to extraction of D N A at neutral pH. Therefore, experiments were performed with dou- ble-stranded and single-stranded D N A in the pres- ence of NaOAc (pH 7.5) and up to 500 mM Mg 2+

At 50 mM MgC12 single-stranded DNA could be completely extracted (Fig. 5A), while all of the double-stranded species remained quantitatively in

A B

f

OC ds linear

relaxed ccc

SS

Fig. 2. Influence of Mg 2 + concentration on the acid-phenol extraction of relaxed ccc DNA. 30 #1 samples containing 0.2/z g of relaxed ccc DNA (and also other DNA species) were acid-phenol extracted under standard conditions except for variations in the Mg '÷ concentration. A: Aliquots of the aqueous phase were analyzed by agarose gel electrophoresis. Mg 2 + concentration: (b), 0, (c), 1.5, (d). 5, (e), 10, (f), 20 and (g) 30 mM. References are shown in lanes a and h. B: The pellet from Expt. g (part A of this figure) was redissolved and analyzed as above (lane a). Lane b contains single-stranded (ss) DNA as reference, ds, double-stranded.

a o .... c . . . . a ~ ¢ e

oc

, linear

laxed ccc

I superhe l i ca l ccc

S S

Fig. 3. Influence of Mg 2+ concentration on the acid-phenol extraction of superhelical DNA. 30 #l-samples containing 0.3 #g of superhelical DNA as well as open circular (oc) and single-stranded (ss) DNA (lane a) were acid-phenol extracted under standard conditions at 0 (lane b) and 2 mM (lane c) MgCI 2. Analysis was performed as described in Fig. 2.

Fig. 4. Test of denaturation of relaxed ccc DNA under extrac- tion conditions using endonuclease S I. Conditions for incuba- tion with S 1 were identical to those for acid-phenol extractions except for the presence of 2 mM ZnSO 4 (for details see Materi- als and Methods). The assays were performed in the presence or absence of both MgC12 and phenol, a, reference; b, no Mg 2+, no phenol; c, no Mg 2+, 8% phenol; d, 30 mM Mg 2+, no phenol; e, 30 mM Mg 2+, no phenol; e, 30 mM Mg 2÷, 8% phenol, oc, open circular; ds, double-stranded.

A B

a d e f g a b c

;d ccc

Fig. 5. Effect of Mg 2+ on DNA extraction at pH 7.5. A. Single-stranded (ss) DNA: 0.l #g of ss DNA were treated under standard conditions except that NaOAc (pH 4.0) was substituted by NaOAc (pH "/.5). Mg 2+ concentration was (b), 0, (c), 10, (d), 20, (e), 30, (f), 40 and (g) 50 mM. The reference is shown in lane a. B. Double-stranded (ds) DNA: 0.4 #g of ds DNA were treated under standard conditions except that NaOAc (pH 4.0) was substituted by NaOAc (pH 7.5). Mg 2+ concentration was (b) 50 and (c) 500 mM. The reference is shown in lane a.

the aqueous phase even at 500 mM MgC12 (Fig. 5B).

Extraction of DNA fragments A 3H-labeled HpalI digest of double-stranded

fd DNA (fragment lengths between 1536 and 6 nucleotides) was subjected to acid-phenol extrac- tion. Less than 3% of the acid-precipitable counts remained in the aqueous phase, and no bands could be detected after polyacrylamide gel electro- phoresis and ethidium bromide staining (data not shown). The smallest fragment visible in the con- trol lane was about 150 N long. The results taken together indicate that at least fragments of chain length greater than 150 N can be extracted. This seems to be different under the conditions em- ployed by Zasloff et al. [1]. These authors reported that non-ccc double-stranded DNA species of about 1500 N or less remain in the aqueous phase.

Behaviour of RNA In contrast to DNA, it seems that RNA cannot

be extracted by the acid-phenol treatment. A 3H- labeled tRNA-mixture remained quantitatively in the water phase even at 100 mM Mg 2+. To test if this finding also holds for other chain lengths a partially degraded mixture of 23, 16 and 5 S rRNA and tRNA was used, which virtually contained all chain lengths up to about 3000 N. All RNA species remained quantitatively in the aqueous layer as verified by gel electrophoresis (data not shown).

D i s c u s s i o n

Our results indicate that the selective extraction of different species of DNA by phenol depends on differences in their hydrophilic characters. At pH 4.0, in the presence of 1.5 mM MgC12, non-ccc DNA is extracted. Under these conditions the DNA is converted into its single-stranded form, which is less hydrophilic than the duplex, as the bases are not shielded from the aqueous environ- ment. Moreover, at this pH a considerable fraction of the bases is protonated [6,7]. This leads to a significant reduction of the over all charge of the DNA.

Mg 2+ is essential for the extraction, but can be replaced by Na +. However, a 100-fold higher c o n -

centration of the latter is required (our un- published results). This indicates that the effect of the salt cannot simply be explained by an increase in the ionic strength of the medium. Probably direct Mg2+-DNA interactions play a role. The difference between the required concentrations of Mg 2+ and Na + agrees with the higher DNA bind- ing constant of Mg 2÷ [8].

Mg2+-DNA association mainly involves the phosphate groups [9], although complex formation with the bases cannot completely he ruled out [10]. Metal ion binding as well as base protonation reduce the over-all charge of the DNA. Both ef- fects together with the exposure of the hydro- phobic bases to the aqueous environment might explain the reduction of the solubility of the DNA in the water phase.

For topological reasons both types of ccc DNA are relatively resistant to complete denaturation. They behave differently, however, as far as local denaturation is concerned: while negatively super- helical DNA possesses single-stranded regions even at pH 4.0 [11-13], relaxed ccc DNA contains no unpaired bases at pH 4.0, as indicated by our results. This difference seems to be reflected in the extraction behaviour of these species.

Superhelical DNA requires only a slightly higher MgC12 concentration for extraction than non-ccc DNA. Therefore, we propose that superhelical DNA exists in a largely denaturated form under our experimental conditions. This form is almost as hydrolbhobic as completely single-stranded DNA. A small increase in charge compensation by Mg 2+ suffices to remove it from the aqueous phase.

Relaxed ccc DNA, on the other hand, is ex- tracted only at MgC12 concentrations greater than 30 mM. It retains its completeley double-stranded structure. The extraction of this species can be explained by the assumption of extensive charge compensation at this Mg 2+ concentration. It should be emphasized that protonation of the duplex seems to be essential for this effect, be- cause extraction of this DNA is not observed at pH 7.5 even in the presence of 500 mM MgCI z. Under these conditions, also non-ccc DNA re- mains in the aqueous phase. Single-stranded DNA, however, is extracted at 50 mM MgCI 2. This is not surprising, since, due to its exposed bases, the single-stranded form is less hydrophilic than the

duplex. Obviously, charge compensation by Mg 2+ alone suffices to crucially increase the affinity of single-stranded DNA for the organic phase.

Unexpectedly, all extracted DNA species were not dissolved in the organic phase, but formed a pellet. At present we cannot give an explanation for this phenomenon.

In contrast to DNA, RNA remains in the aque- ous phase even at pH 4.0 and 100 mM MgC12. We propose that, in the case of RNA interactions of the phosphate anions with metal ions are pre- vented or at least strongly reduced under the con- ditions employed. Bolton and Kearns [14] present experimental evidence that the 2'-OH groups in RNA are hydrogen bonded to water molecules, which are simultaneously hydrogen bonded to the 3'-phosphate groups. This hydrogen bonding scheme could account for a reduced binding of Mg 2+ ions to RNA.

Acknowledgments

We wish to thank W. Guschlbauer for reading the manuscript and the Deutsche Forschungs- gemeinschaft and the Bundesminister fiir Wissen- schaft und Technologic for financial support.

References

1 Zasloff, M., Ginder, G.D. and Felsenfeld, G. (1978) Nucleic Acids Res. 5, 1139-1152

2 Hofer, B. and K6ster, H. (1979) FEBS Lett. 102, 87-90 3 Hofer, B. and K/Sster, H. (1980) Nucleic Acids Res. 8,

6143-6162 4 Tabak, H.F. and Flavell, R.A. (1978) Nucleic Acids Res. 5,

2321-2332 5 Blagoy, Yu.P., Sorokin, V.A., Valeev, V.A. Khomenko, S.A.

and Gladchenko, G.O. (1977) Mol. Biol. 11,748-756 6 Courtais, Y., Fromageot, P. and Guschlbauer, W. (1968)

Eur. J. Biochem. 6, 493-501 7 Hermann, P. and Fredericq, E. (1977) Nucleic Acids Res. 4,

29398b12947 8 Sander, C. and Ts'o, P.O.P. (1971) J. Mol. Biol. 55, 1-12 9 Pezzano, H. and Podo, F. (1980) Chem. Rev. 80, 365-401

10 Reuben, J. and Gabbay, E.J. (1975) Biochemistry 14, 1230-1235

II Law, P.P. and Gray, H.B., Jr. (1979) Nucleic Acids Res. 6, 331-357

12 Dasgupta, S., Allison, D.P., Snyder, C.E. and Mitra, S. (1977) J. Biol. Chem. 252, 5916-5923

13 Benham, C.J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3870-3874

14 Bolton, P. and Kearns, D. (1979) J. Am. Chem. Soc. 101, 479-484