Biochimica et Biophysica Acta, 740 (1983) !-7
Elsevier
1
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: D N A 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 described [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 circular; superhelical closed circular; relaxed closed
circular) we gained some insight into the mechanism 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 singlestranded 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 described. Double-stranded fd DNAs were 3Hlabeled 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 described 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 procedure 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. (Deviations 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 residual 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 potential 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 electrophoresed in vertical or horizontal slab gels of
1% agarose in 40 mM Tris-HOAc/20 mM
N a O A c / l mM E D T A / 0 . 5 / t g / m l ethidium
bromide (pH 7.7) [4] at 80-100 V for 2-6 h. Visualization 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 phenol. 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 u r e a / 2 0 mM EDTA/0.05%
xylene-cyanol/0.05% Bromophenol blue) and the
products were analyzed by agarose gel electrophoresis (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 containing 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 linear
relaxed
ccc
ss l i n e a r
ss c i r c u l a r
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-phenolextraction' protocoldescribed in Materials and Methods, but with the
modificationsgivenbelow. Aliquotsof the aqueous phases were
analyzed by agarose gel electrophoresis. Lanes a and g: references of double-strandedand single-stranded(ss) DNA species.
respectively. Extraction procedure with the complete system
(b), without Mg2+ (c), without NaOAc, pH 4.0 (d), without
phenol (e), without phenolicphase, but with a phenol-saturated
aqueous phase (f). oc, open circular.
adjusted to 1.5 mM MgC12, and acidified by addition of NaOAc (pH 4.0). Phenol was added, the
mixture was vortexed and centrifuged. The aqueous phase was withdrawn and analyzed by liquid
scintillation counting and agarose gel electrophoresis (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
D N A 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
D N A 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 D N A had vanished, while
two new bands with the mobilities of singlestranded unit length linear and circular DNA had
appeared. (As 90% of the radioactivity had remained 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.
!
2
3
4
5
6
Components of the system
DNA
DNA/MgCI 2/NaOAc (pH 4)phenol
DNA/2qaOAc (pH 4)/phenol
DNA/MgC12/phenol
DNA/MgCI 2,/NaOAc (pH 4)
DNA/MgCI2/NaOAc (pH 4)/phenol
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 performed 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 remained in solution. Non-ccc species were denatured 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 p H 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
Components in aqueous phase after extraction procedure
cpm
(%)
DNA species
see
Fig. 1
100
53
90
95
100
86
ccc, non-ccc
ccc
ccc, ss
ccc, non-ccc
ccc, non-ccc
ccc, ss
lane a
lane b
lane c
lane d
lane e
lane f
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 concentrations
Earlier experiments had demonstrated that
M g C I : concentrations substantially greater than
1.5 m M 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
m M (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 m M Mg 2+
(Fig. 3). U p to 1.5 m M Mg 2÷ most of this D N A
remained in the aqueous phase (data not shown).
The Mg 2+ concentrations necessary for complete extraction of different D N A species are summarized 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
M I N I M A L Mg 2+ C O N C E N T R A T I O N S FOR COMPLETE
EXTRACTION U N D E R S T A N D A R D C O N D I T I O N S
D N A species
Mg 2 + concentration
(mM)
Non-ccc (fd)
Superhelical-ccc ( f d )
Relaxed-ccc ( f d )
1.0- 1.5
1.5- 2.0
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 m M 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 D N A s were completely degraded. This
proves that the enzyme is active under these conditions and provides further evidence for the denaturation of these D N A species. The relaxed ccc
DNA, on the other hand, remained completely
intact even at 30 m M MgCI2.
In control experiments without phenol it was
observed that S 1 converts open circular D N A 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 p H 7. 5
The finding that relaxed ccc D N A can be extracted without denaturation at 30 mM Mg 2÷
raised the question, if elevated MgCI 2 concentrations can lead to extraction of D N A at neutral pH.
Therefore, experiments were performed with double-stranded and single-stranded D N A in the presence of N a O A c (pH 7.5) and up to 500 mM Mg 2+
At 50 m M MgC12 single-stranded D N A 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 D N A (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
, linear
laxed ccc
oc
I superhelical
ccc
SS
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 extraction conditions using endonuclease S I. Conditions for incubation with S 1 were identical to those for acid-phenol extractions
except for the presence of 2 mM ZnSO 4 (for details see Materials 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
a
d
B
e
f
g
a
b
c
;d ccc
Fig. 5. Effect of Mg 2+ on DNA extraction at p H 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 extraction. Less than 3% of the acid-precipitable counts
remained in the aqueous phase, and no bands
could be detected after polyacrylamide gel electrophoresis and ethidium bromide staining (data not
shown). The smallest fragment visible in the control 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 employed 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 3Hlabeled 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).
Discussion
Our results indicate that the selective extraction
of different species of D N A by phenol depends on
differences in their hydrophilic characters. At pH
4.0, in the presence of 1.5 mM MgC12, non-ccc
D N A is extracted. Under these conditions the
D N A is converted into its single-stranded form,
which is less hydrophilic than the duplex, as the
bases are not shielded from the aqueous environment. 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 unpublished 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 binding 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 effects together with the exposure of the hydrophobic 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 superhelical D N A 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
D N A 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 extracted 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, because extraction of this DNA is not observed at
p H 7.5 even in the presence of 500 mM MgCI z.
Under these conditions, also non-ccc DNA remains 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 aqueous 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 prevented or at least strongly reduced under the conditions 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 Forschungsgemeinschaft and the Bundesminister fiir Wissenschaft und Technologic for financial support.
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