You are on page 1of 8

ANALYTICAL BIOCHEMJSTRY 77, 332-339 (1977)

Electrophoresis of Denatured DNA in Gels


Containing Formaldehyde

M. YANEVA, J. MLADENOVA, AND G. DESSEV


Institute of Biochemistry, Bulgarian Academy of Sciences, Sofia 13, Bulgaria

Received April 26, 1976; Accepted July 2, 1976

The possibility of using fomaldehyde as a denaturant for DNA in agarose- or


polyacrylamide-gel electrophoretic systems was studied. It was found possible to
fractionate and determine the molecular weights of denatured DNA using
calibration curves constructed with single-stranded DNA fragments of known
molecular weights.

INTRODUCTION
Formaldehyde was first applied as a denaturing agent in a polyacryl-
amide-gel electrophoretic system by Boedtker (l), who tried to achieve
a conformation-independent molecular weight determination of RNA.
Later, this method was criticized by Staynov et al. (2) on the basis of
a possible cross-linking of RNA by formaldehyde. These authors proposed
a method for the determination of the molecular weight of RNA by electro-
phoresis in polyacrylamide gels containing formamide, which has also
been applied to DNA fractionation (3,4).
In this work, we investigate the possibility of denaturing and electro-
phoresing DNA in polyacrylamide or agarose gels in the presence of
formaldehyde, using restriction nuclease DNA fragments as standards.

MATERIALS AND METHODS


All reagents used were analytical grade. Agarose was from Koch-Light.
Formaldehyde (Merck, 36%) was neutralized to pH 7.0 with NaHCO,
and used as 33% solution. Acrylamide was recrystallized from chloroform
and N,N’-methylene-bisacrylamide was recrystallized from acetone.
Samples of bacteriophage PM2 DNA fragments of known molecular
weights, obtained by digestion with restriction nuclease III from Hemo-
phifus in&wvzzae (43, were generous gifts from Dr. Zachau (University
of Mtinchen) and Dr. Van Holde (University of Oregon).
Ribosomal RNA fromEscherichia coli, rat liver, and yeast was isolated
using routine methods. The molecular weights of the RNAs used in this
work were those given by Attardi and Amaldi (6).
Slabs of agarose gels (1.5%, containing 20 mM sodium phosphate buffer,
pH 7.0) were prepared as described elsewhere (7). To prepare the 2.5%

332
Copyright 0 1977 by Academic Press, Inc.
All rights of reproduction m any form reserved. ISSN 0003-2697
ELECTROPHORESIS OF DENATURED DNA 333

10
1 2 3 4 5 6 7 8 9 10 11 12
DISTANCE MIGRATED (CM)

FIG. 1. Log of chain length versus mobility plot of bacteriophage PM2 single-stranded
DNA fragments, obtained with Hemophilus injfuenzae restriction nuclease III, in a 2.5%
polyacrylamide gel containing formaldehyde. Closed circles, fragments isolated in the
laboratory of Dr. Zachau; open circles, fragments isolated in the laboratory of Dr. Van
Holde.

or 7.5% polyacrylamide gels, mixtures containing 2.5% (7.5%) acrylamide,


0.125% (0.365%) N,N’-methylene-bisacrylamide, 20 mM sodium phos-
phate buffer, pH 7.0, and 3% formaldehyde were polymerized at room
temperature in ldcm-long, 7-mm-i.d. glass tubes by addition ofN,N,N’,-
N’-tetramethylethylenediamine (TEMED) (46 pl/lOO ml) and ammonium
persulfate (final concentration, 0.087%). The solution was overlaid with
water. For both agarose and polyacrylamide gels, the electrode vessels
contained 20 mM sodium phosphate buffer, pH 7.0.
DNA or RNA samples containing 0.05-o. 1 AzGOunit in 0.1 ml of 2 mM
sodium phosphate buffer (pH 7.0)-0.01 mM EDTA were made 3% in
formaldehyde, heated 3 min in a boiling-water bath (96”C), cooled, and
applied directly to the gels (agarose) or applied to the gels (polyacrylamide)
after addition of sucrose to 6% and bromophenol blue to 0.001%. In some
cases tRNA or 5s RNA from rat liver was added as a mobility marker.
334 YANEVA, MLADENOVA AND DESSEV

104
3 4 5 6 I 8 9
DISTANCE MIGRATED (Ct.41

FIG. 2. Log of chain length versus mobility plot of the same fragments as in Fig. 1
and high molecular weight ribosomal RNAs from different species, electrophoresed in a 1.5%
agarose gel containing formaldehyde. Symbols as in Fig. 1.

Electrophoresis was carried out at room temperature at about 8 V/cm for


2.5 hr (agarose) and at 1 V/cm for 20 hr (polyacrylamide). After the
run, the gels were stained overnight with Stains All (8), and destained by
exposure to visible light, and the distances traveled by the fractions were
measured directly or after scanning at 550 nm in a spectrodensitometer (9).
Isolation of “structured” chromatin from Guerin ascites tumor,
digestion with micrococcal nuclease (EC 3.1.4.7) and deoxyribonuclease
I (EC 3.1.4.5), and isolation of DNA were performed as described else-
where. (10).

RESULTS AND DISCUSSION


It is well known that heating in the presence of formaldehyde causes
a complete and irreversible denaturation of DNA (11). Hayward and Smith
(12) have used NaOH to denature DNA prior to electrophoresis in agarose
gels. However, it is well known that the highly repeated sequences in
mammalian DNA rapidly renature after removal of the denaturing agent
(13). In our gels, formaldehyde is present in the gel throughout the run,
which rules out the possibility of renaturation.
Although heating may cause chain scissions in RNA (14)) electrophore-
tically we found very little RNA degradation under our conditions of
ELECTROPHORESIS OF DENATURED DNA 335

I
2 4 6 0 10 12 14
DISTANCE MIGRATED (CM)

FIG. 3. Electrophoretic profile in a 2.5% polyacrylamide gel containing formaldehyde of


denatured DNA fragments isolated from structured chomatin digested with micrococcal
nuclease to 9% acid-soluble products under conditions described elsewhere (10). The
numbers of the fractions correspond to those in Table 1.

denaturation, provided measures were taken to avoid ribonuclease action


during the isolation of RNA. On the other hand, there is a small amount
of hyperchromicity developing above 80°C in rat liver ribosomal RNA
(but not in RNA from yeast or E. co/i) which is due to the melting of
some GC-rich regions (15). As a rule, RNA should be heated at the
lowest temperature at which its denaturation is complete.
Bromphenol blue, or any other dye of low molecular weight, is not
the ideal mobility marker, because even in concentrated gels it gives a
rather broad and diffuse band. Much better results are obtained with
tRNA or 5s RNA or a mixture of the two as reference substances,
provided they do not overlap with the DNA fractions. When they did,
it was found useful to run two samples in parallel, one with the marker
and another without it, so that one could find the position of the marker
on the second gel. Bromphenol blue is washed out during the soaking
of the gel in water prior to the staining, while tRNA and 5s RNA remain
in the gel. This becomes particularly important with dilute gels, which
increase their length during staining.
Figures 1 and 2 show the molecular weight versus mobility plots of
denatured PM2 DNA fragments in 2.5% polyacrylamide and 1.5% agarose
formaldehyde-containing gels, respectively. It is seen that the relationship
is linear over a wide range of molecular weights, with certain nonlinear
regions. No change in the number of fragments occurs after denaturation,
compared to their number under nondenaturing conditions. Therefore, no
intermolecular cross-links are formed.
To determine whether the denaturation procedure caused intra-
molecular cross-linking in the single-stranded RNA, we tried to detect
methylene-bis-dinucleotides in an alkaline hydrolysate (0.5 NKOH, 18 hr,
37°C) of rat liver ribosomal RNA treated for 3 min at 96°C with 3%
336 YANEVA, MLADENOVA AND DESSEV

2 4 6 a 10 12 14
DISTANCE MIGRATED (CM)

FIG. 4. Electrophoretic profile of single-stranded DNA fragments from a deoxyribo-


nulcease I digest of structured chomatin. Two Alo,, units of chromatin in 1 ml containing
2 mM TES buffer (pH 7.8), 5 PM EDTA, and 100 PM MgCl, were digested for 1 hr at
37°C with 2 pg of deoxyribonuclease I (Worthington) to 18.5% acid-soluble products. DNA
was isolated, denatured, and electrophoresed in a 7.5% polyacrylamide gel containing formal-
dehyde. The numbers on the peaks show their chain length in nucleotides.

formaldehyde. The hydrolysate was analyzed on a Dowex l-X4 column


(1 x 4 cm formate form) exactly as described by Feldman (16). Unfixed
RNA was processed in the same way as control. No significant difference
was detected between the fixed and the control RNA in the region where
dinucleotides are eluted (3.6 M HCOOH). Taking into account the sensi-
tivity of the procedure, the proportions of dinucleotides could not be
above l%, in agreement with McGhee and von Hippel(17) who concluded
that formation of cross-linked methylene dinucleotides is completely
negligible in the reaction between formaldehyde and deoxyribonucleoside
monophosphates.
Under the present conditions, the high molecular weight ribosomal
RNAs exhibit mobilities higher than those of DNA fragments of equal
size. All points, however, fall on a line almost parallel to that of DNA.
Using the same DNA fragments, calibration curves can be constructed
for gels of different concentrations (data not shown). In this way the
linear part of the plot can be extended to cover a molecular weight range
up to at least 3.25 x lo6 daltons.
The following experiments illustrate the possibility of application of
the method to DNA fragments of different origins.
Figure 3 shows the electrophoretic profile of denatured DNA fragments
isolated from structured chromatin digested with micrococcal nuclease,
as described elsewhere (10). Examination of the same sample by electro-
phoresis under nondenaturing conditions revealed a series of fragments
whose molecular weights were integral multiples of 180 base pairs (IO),
as also found for digests of whole nuclei (4). When denatured and electro-
phoresed under the present conditions, such samples were found to consist
of discrete single-stranded DNA fragments (Fig. 3), whose molecular
weights are given in Table 1. All fragments are 40- 60 nucleotides shorter
ELECTROPHORESIS OF DENATURED DNA 337

TABLE I

AVERAGE MOLECULAR WEIGHTSOFTHE SINGLE-STRANDED DNA FRACTIONS IN F1c.3,


DETERMINED USING THE CALIBRATION CURVE IN FIG. 1

Nucleotides Nucleotides
Fraction determined calculated”
number (a) (b) b-a

1 140 180 40
2 305 360 55
3 500 540 40
4 680 720 40
5 860 900 40
6 1020 1080 60
7 1200 1260 60

n On the bases of an IlO-base pair repeating unit (4, 10).

than those calculated on the basis of an 180-base pair repeat. The difference
probably reflects the more advanced digestion of the “spacer” DNA,
which has been estimated to about 40 base pairs on the average (4).
Indeed, Fig. 3 shows the presence of some amount of DNA moving faster
than the “monomer.”
Another example is given in Fig. 4, which presents the electrophoretic

200

100
90
G 80
E 70
F 60
zd 50
z 40

10
3 4 5 6 7 8 9 10 11 12 13
DISTANCE MIGRATED (CM)

FIG. 5. Log of chain length versus mobility plot of the fragments in Fig. 4. The chain
lengths are assigned by analogy with the values given by No11 (3).
338 YANEVA, MLADENOVA AND DESSEV

profile, in a 7.5% polyacrylamide gel containing formaldehyde, of a DNase


I digest of structured chromatin. The profile reveals a series of fragments
of DNA. Similar results have been obtained by No11 (3) with DNase digests
of whole rat liver nuclei. The sizes of these fragments, precisely deter-
mined by comparison with sequenced DNA fragments, have been found
to be multiples of lo-nucleotide repeating units.
Our pattern (Fig. 4) is strikingly similar to that published by No11 (3)
and exhibits the same characteristic pattern with strong bands at 80 and
110 nucleotides and relatively weak bands at 100 and 130 nucleotides.
These fractions may be used to construct a calibration curve for 7.5%
gels containing formaldehyde, which is shown in Fig. 5. The mobilities
of 5s RNA from rat liver, tRNA Phe from yeast, and the 3’-half of the
latter (120,76, and 39 nucleotides, respectively) in this system were higher
than the mobilities of DNA fragments of the same size. This is consistent
with the above-mentioned behavior of the ribosomal RNAs and also with
the observation of No11 concerning the mobility of 5.8s RNA from yeast
and larger RNA fragments in the formamide-polyacrylamide-gel
system (3).
The reaction of DNA with formaldehyde is a subject of numerous
studies (for example see 11, 17, and 18) and is by no means a simple
process. Whatever the changes, however, in DNA reacted with formal-
dehyde, our results demonstrate that the present method may be used
for molecular weight determinations of single-stranded DNA fragments,
using standards of known molecular weight. The method represents an
alternative to the electrophoresis in a nonaqueous medium (2) and, in our
hands, has been proven to be technically easier. An important advantage
of formaldehyde over formamide is that the former can be used with
agarose and possibly with mixed agarose-polyacrylamide gels, which do
not set in the presence of formamide.

ACKNOWLEDGMENTS
We wish to thank Dr. Zachau from the University of Miinchen and Dr. Van Holde
from the University of Oregon for the generous gifts of fragments of PM2 DNA.

REFERENCES
1. Boedtker, H. (1971) Biochim. Biophys. Acrn 240,448-453.
2. Staynov, D. Z., Pinder, J. C., and Gratzer, W. B. (1972)NutureNew Biol. 235,108- 110.
3. Noll, M. (1974) Nucleic Acids Res. 1, 1573-1578.
4. Van Holde, K. E., Shaw, B. R., Lohr, D., Herman, T. M., and Kovacic, R. T. (1975)
Proceedings of the Tenth FEBS Meeting, Paris, pp. 57-72.
5. Streek, R. E., Philippsen, P., and Zachau, H. G. (1974) Eur. J. Biochem. 45, 489-499.
6. Attardi, G., and Amaldi, F. (1970) Annu. Rev. Biochem. 39, 183-226.
7. Tsanev, R. G. (1965) Biochim. Biophys. AC&I 103, 374-382.
8. Philippsen, P., and Zachau, H. G. (1972) Biochim. Biophys. Acta 277, 623-628.
9. Tsanev, R. G., and Staynov, D. Z. (1964) Biokhimiu 29, 1126- 1131.
ELECTROPHORESIS OF DENATURED DNA 339

10. Yaneva, M., and Dessev, G. (1976) Eur. .I. Biochern. 66, 535-542.
11. Feldman, M. Ya. (1973) Progr. Nucleic Acid Res. Mol. Biol. 13, l-49.
12. Hayward, G. S., and Smith, M. G. (1972) J. Mol. Biol. 63, 383-395.
13. Britten, R. J., and Kohne, D. E. (1968) Science 161, 529-540.
14. Brown, D. M. (1974) in Basic Principles in Nucleic Acid Chemistry (Ts’o, P. 0. P.,
ed.), Vol. II, p. 53, Academic Press, New York; and the references quoted therein.
15. Grancharov, K. Ch., Staynov, D. Z., and Dessev, G. N. (1974) Acrn Biol. Med.
Ger. 33, 725-731.
16. Feldman, M. Ya. (1967) Biochim. Biophys. Actn 149, 20-34.
17. McGhee, J. D., and von Hippel, P. H. (1975) Biochemistry 14, 1281-1296.
18. McGhee, J. D., and von Hippel, P. H. (1975) Biochemistry 14, 1297-1303.