Download Gel electrophoresis of partially denatured DNA. Retardation effect

volume 10 Number 151982
Nucleic Acids Research
Gel electrophoresis of partially denatured DNA. Retardation effect: its analysis and application
V.I.Lyamichev, I.G.Panyutin and Yu.L.Lyubchenko
Institute of Molecular Genetics, USSR Academy of Sciences, Moscow, USSR
Received 6 May 1982; Revised and Accepted 9 July 1982
ABSTRACT
The hypothesis about the role of partial denaturation in
DNA retardation during its electrophoresis in denaturing gel
/1,2/ was tested. We used partially melted DNA molecules in
which the size of the melted regions and their location were
known. They were obtained through glyoxal treatment of the melted regions by a procedure allowing the denatured state to be
fixed at any point within the melting range. The approach and
the availability of the melting maps of DNAs made it possible
to investigate DNA molecules differing in length and in the
size of the melted regions. The presence of a denatured region
at the end of the molecule or inside of it was shown to decrease its electrophoretic mobility, the effect depending on
the size of the melted region and on the DNA length. On the
basis of the experimental results an explanation is proposed
for the cause of retardation in the oase of partially denatured DNA.
INTRODUCTION
Various chromatographic techniques and gel electrophoresis are widely used for length-wise separation of DNA. Yet,
until recently there have been no methods for separation DNA
fragments in accordance with their base sequence, whereas such
methods would allow a two-dimensional analysis of DNA like the
electrophoresis - isoelectric focusing for proteins. In the
past two years two techniques have been proposed for the sequence-wise fractionation of DNA. The technique described in
/3/ involves the use of intercalated Hoechst dye with a covalently bound polyethylene glycol molecule. The AT specificity
of the dye makes for its preferential binding to the AT-enriched DNA fragments, while the presence of bulky polyethylene
glycol molecules decreases the electrophoretic mobility of the
DNA fragments in proportion to the number of dye molecules
O IR L Prats Limited, Oxford, England.
0306-1048/82/1015-48133 2.00/0
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bound. The use of the Hoechst dye makes it possible, in the
authors» estimation /3/» to separate DNAs having a 1% difference in the AT content. Fischer and Lerman /1,2/ have discovered a sharp fall of the mobility of DNA in the case of its
partial denaturation. Their experiments were carried out in a
4% polyacrylamide gel containing a concentration gradient of
the denaturing agents at 60°C. In the course of its migration
in the gel the DNA fell under conditions corresponding to the
beginning of its melting, whereupon its mobility was sharply
decreased. The same happened to restriction fragments of A
phage DNA: the AT-enriched sections were the first to come to
a halt in electrophoresis. The length-wise separation of DNA
restriction fragments and subsequent fractination under denaturing conditions yielded 350 fractions for EooR1 fragments
of E.coli DHA /1/.
This may be a convenient approach for two-dimensional
fractionation of DHA. However, the data presented in the papers
/1,2/ do not definitively explain why the retardation effect
is observed. Nor is it clear how the retardation efficiency
depends on the size of the denatured section or whether the
effect depends on the terminal or internal location of the
unwound region. The present study tackles these questions and
some others too.
We used partially melted DNA molecules in which the size
of the melted regions and their location were known. They were
obtained through glyoxal treatment of the melted regions by a
procedure allowing the denatured state to be fixed at any point
within the melting range /4,5/. Because of this it had been possible to construct electron-microscopic melting maps for a number of phage and plasmid DNAs /5»6/. The approach and the
availability of the melting maps made it possible to investigate DNA molecules differing in length and in the size and location of the melted regions. The emergence of one unwound region in any part of the molecule proved sufficient to slow
down the mobility of DNA in a gel; a melted region of 42O-5OObp
practically halts the DNA molecule at the start. On the basis
of the experimental results an explanation is proposed for the
retardation effect in the oase of partially denatured DNA. We
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also discuss some advantages of the sequence-wise fractionation
procedure Involving glyoial fixation of the melted regions and
possible applications.
MATERIALS AND METHODS
We used the DNA of plasmids pAO3 (the E.ooli C600 strain
with this plasmid was kindly supplied by Dr.A.Oka) and pBR322
extracted and purified by Dr. L.V.Neumyvakin in this laboratory as described in /5/. ColE1 plasmid DNA and the C1 fragment
produced by Bspl cleavage of T7 DHA / 7 / were made available by
Dr.A.H.Rekesh from this Institute and Dr.E.P.Zaichikov from
the Novosibirsk Institute of Organic Chemistry, USSR Academy of
Sciences (Siberian Branch) respectively. DNA melting was performed on a Cary 219 double-beam recording spectrophotometer
(Varian, Switzerland) equipped with especially designed thermostatically controlled cell, with the temperature changing continuously at a constant rate of 0,1°C per minute. The melting
curves were differentiated as in /5/ by means of an HP 9825A
calculator (Hewlett - Packard, USA) interfaced with a HP 9864A
digitizer.
The DNA molecules were fixed with glyoxal in a preset
partially denatured Btate as described in /4,5/.
DNA electrophoresis was carried out in a 4% polyacrylamlde gel (0,1% bisacrylamide) containing denaturing agents
formamide (Serva) and urea in concentrations bringing down the
DNA melting temperature by the value of 0°C to 40°C. The buffer
solution used for electrophoresis contained 40 mM Tris-HCl
(pH 7.8), 5 mM sodium acetate and 1 mM EDTA. Electrophoresis
was performed at room temperature (~25°C) with continuous
pumping of the buffer solution. 2 0 ^ 1 of DNA (25>tg/ml) with
glyoxal-fixed denatured regions in O.ixSSC were applied to
slab gel cells and electrophoresis was performed for about
15 hrs at a field intensity of 3-4 v/cm. After the completion
of electrophoresis the slabs were placed for 30 mln into a
solution of 1/Mg/ml ethidlum bromide, then the gels were photographed through a red filter.
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RESULTS
1. Baaic approach. To investigate the retardation of partially denatured DNA in a gel we assayed the mobility of DNAs
with unwound regions of known size and location. Such DNA preparations were obtained through glyoxal fixation of unwound
regions within the melting range /4/. The technique makes it
possible to fix only those regions melted at a chosen temperature in the melting range. The prooedure can be performed at
different temperatures, and subsequent electron-microscopic
analysis allows all regions melted at a given temperature to
be mapped. In this way the melting of ColE1 DNA /5/ and pBR322
DNA /6/ was visualized.
Figure 1 shows the differential (a) and the integral (b)
melting curves for EcoRI-cleft pAO3 plasmid DNA, which is one
fourth (1683 bp) of ColE1 DNA /9/. The first two peaks in
curve (a) correspond t o ~ 5 % and~20% denaturation of pA03 DNA
respectively. Electron-microscopic analysis of DNA preparations
fixed at 62°C and 65°C (both temperatures lying beyond the two
peaks) shows them to be due to the melting of DNA regions of
-^70 and 420 bp long from the same end of DNA /5 t 8/. ffe used
FIGURE 1. Differential (a) and integral (b) melting curves for
pA03 DNA in O.ixSSC. Arrows indicate fixation temperatures corresponding to pA03(1) and pAO3(2) DNA's respectively (see Figure 2 ) .
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these preparations of pA03 DNA for studying the retardation
effect. Besides, we used similarly obtained preparations of the
C1 fragment of T7 DNA (1461 bp) produced by Bspl. Melting maps
of this DNA will be published elsewhere. All the DNA preparations used in the present study are diagrammatically shown in
Figure 2. The shaded area in each diagram schematically denotes the size of the melted region. This particular selection
of DNA samples allows a study of molecules with denatured regions varying from ~70 to ~ 500 bp.
2. The varying size of the denatured region. Figure 3 presents
the results of electrophoresis for a series of C1 DNA preparations. As the size of the denatured region grows the mobility
of DNA decreases. The denaturation of a region ~500 bp long
halts the DNA molecules at the start (Figure 3d). The effect
is qualitatively the same for pA03 DNA (Figure 4a,b,c). While
the melting of 420 bp almost completely stops pA03 DNA, that
of 70 bp only slightly changes its mobility.
Thus, glyoxal fixation of the melted regions causes the
mobility of DNA molecules to decrease; the larger the melted
region the stronger the effect.
a) I
130 bp
V////A
I
CUD DNA
1
C1 (21 DNA
1
C1(3) DNA
210 bp
b) I
V////////A
500 bp
c) V///////////W////////A
70bp
d) V/A
I pA03(1) DNA
i20bp
e) V///////////////////A
I pA03(2) DNA
0
500
1000
Length in base pairs
1500
FIGURE 2. Diagrammatic representation of the types of molecules
obtained by fixing partially denatured states. Shaded areas
show the position and size of fixed regions. Clear areas stand
for native DNA sections, a) C1(1) DNA contains 130-bp fixed
region; b) C1(2) DNA - 210 bp: c) C1(3) DNA - 500 bp;
d) pA03(D - 70 bp; e) pA03(2) DNA - 420 bp.
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FIGURE 3. Electrophoretic separation of
fixed and native C1 DNA. a) native C1 DNA;
b) C1(1) DNA ( t h i s preparation contained a
small amount of the native molecules);
c) C1(2) DNA; d) C1(3)DNA; Gel contained 35%
formamide, 6M u r e a .
c
a
d
FIGURE 4. Electrophoresis of fixed and
native pAO3 DNA. a) native pAO3 DNA;
b) pA03(1) DNA; c) pAO3(2) DNA. Gel contained 35% formamide, 6M u r e a .
a
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3. The effect of the denaturing agents on the mobility of
glyoxal-fixed DNA. Figures 3 and 4 present the results of experiments in which the denaturing agent in the gel (formamide
with urea) effectively brings down the DNA melting temperature
by 35°C /10/. In this situation the glyoxal-fixed regions are
completely melted /4,5/.
Figures 5 shows the results of electrophoretic separation of C1 DNA, C1 (1) DNA and C1 (2) DNA at a concentration
of the denaturing agents lowering the melting temperature by
20°C (Fig.5). The same experiments were made at different concentrations of denaturing agents (data not shown). These experiments demonstrated that as the concentration of the denaturing agents grew the bands got narrower (see Fig. 3»5) but
did not change any further if the denaturant concentration
continued growing.
The melting of fixed DNA preparations provides an explanation of these results. Figure 6 presents the melting curve for
C1 (2) DNA. The broken line shows the melting curve of C1 DNA
for comparison. The melting pattern can be seen to change after
C1 DNA is fixed with denatured region of 210 bp (see Figure 2b);
this state of DNA corresponds to 74°C and is indicated by the
FIGURE 5. Electrophoresis of native and fixed
formamide and
C1 DNA in a gel
j ~ containing 20%
"^ '
3.5M urea (T
(T eff =45 C ) . a) 01 DNA; b) C1(1) DNA;
c)C1(2) DNA.
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25
30
35
40
45 50
55
60
65
70
75 80
TEMPERRTURE/"C
FIGURE 6. Melting curves for C1 DNA (
) and C1(2) DNA (
In electrophoretic buffer. Arrows (from left to right) Indicate temperatures corresponding conditions of electrophoresis
for C1 (2) DNA.
)
vertical broken line in Figure 6. The melting occurs in the
temperature range from ~25°C to ^ 55°C The addition of denaturing agents lowering the DNA melting temperature /10/ is tantamount to a shift to the right along the DNA melting curve.
Thus different concentrations of formamide with urea in the
gel correspond to different temperature points in the DNA melting curve. As the denaturant concentration grows the glyoxal-fixed regions gradually melt and become completely melted
when a plateau is reached at 55°C. Electrophoretically such
DNA melting is accompanied (Fig.3) by a gradual narrowing of
the band and the formation of a clear-cut enough band closer
to the start, corresponding to the complete melting of the
fixed region.
4. The influence of the position of the unwound region upon
the retardation effect. To find out whether the position of
the unwound DNA region (terminal or internal) has any influence
on the efficiency of retardation, we studied ColE1 DNA cleft
by single-site restriction endonucleases EcoRI and Smal. It
follows from /5,8/ that these DNAs start melting from one and
the same region about 420 bp long, only in the case of EcoRI
it is located at the end of the molecule while in the other
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case it is inside the molecule. Figure 7 shows the results
of these assays. The 420-bp-long unwound region stops both
types of molecules in the same way.
5. The effect of the molecule length. We checked this effect
in the case of ColE1 and pAO3 plaamid DNAs. The latter is one
fourth of the former. A left-hand terminal region of about
70 bp was fixed in both DNAs (see Pigure 2d; ColBI DNA is a
mere continuation of the same molecule to the right; it is
known from / 8 / that this is the lowest-melting region in both
DNAs). Pigure 8 ahows the results of such electrophoretic
assays. While in the case of pA03 (1) DNA the 70-bp-long unwound region decreases its electrophoretic mobility (the diffuse band above the pA03 DBA band), the same unwound region
fails to hold back ColE1 DNA.
6. Application of the retardation effect to denaturation
Figure 9 presents the results of electrophoretic
separation of Bsp I-restriction fragments of native pBR322 DNA
(Pigure 9a) and of this DNA with glyoxal-fixed regions
(Pig. 9b) corresponding to two peaks in the differential melPIGDHE 7. Electrophoresis of fixed ColBI DNA
cut by restriction endonucleases Smal (a)
and EcoRI (b); o) native ColE1 DNA linearized
by EcoRI. A% polyacrylamide gel with 3556
formamide and 6M urea ( 1 . ^= 6 0 0 ) ,
eff
a
b
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FIGURE 8. Photograph of a gel
containing pA03 DNA and ColE1 DNA.
Both DNAs had been c l e f t by EcoRI
and in both the same low-melting
region at the left-hand end of the
molecule (see Figure 2) was fixed.
Polyacrylamide gel with 30%
formamide and 5.3M urea (Teff=55°C).
a) pA03 DBA; b) pA03 (1) DNA;
c) ColE1 DNA; d) fixed ColE1 DBA.
a
FIGURE 9. Electrophoresis of Bspl-cleft
pBR322 DNA without formamide and urea at 25 C
a) native DNA; b) glyoxal-fixed DNA.
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ting curve /6/. One can Bee that fixation leads to a disappearance of bands of fragments A and D due to their retardation.
Hence it is these fragments that contain the fixed unwound
regions. The cleavage map is known for pBR322 DNA, hence it
is possible to localize the sites corresponding to the early
melting of pBR322 DNA. This very localization had been presumed on the basis of electron-mioroscopic mapping of the
latter stages in the melting of pBR322 DBA /6/. Note that as
the DNA fragments containing denatured regions are revealed
due to dlssappearance of corresponding bands in the electrophoretic pattern because of their diffused character and these
bands become more and more diffused as the denaturant concentration falls (see Pigs. 3,5) it is no need to use the denaturant
in gels at all.
DISCUSSION
The results directly demonstrate that the presence of an
unwound region in DNA decrease its electrophoretic mobility.
When the denatured is 42O-5OObp long the DNA is practically
prevented from entering the gel. Why does it happen?
One might reason that the effect is basically due to the
terminal melted regions which gel tangled in the gel and hamper the molecule's movement. Yet, this explanation contradicts the data in Figure 7 which show that a melted region of
420 bp produces the same retardation effect on a linear ColE1
DNA molecule whether terminally or internally located.
In our opinion the most likely cause of the retardation
effect is the thickening of DNA at the site of its local denaturation. Indeed, all experiments were carried out with the
same concentration of acrylamide (4%) and bisacrylamide (0.1%).
According to /11/ the average pore size In such a gel (measured by the mobility of globular proteins) is about 20-30 1,
i.e. only slightly larger than the diameter of the DNA helix
(20 ! ) . Therefore even a slight local swelling of DNA should
hamper its migration. In the case of the Hoechst dye with polyethylene glycol /3/ the latter might do the thickening. In
partially denatured DNA the movement is hampered by coils
forming in the unwound regions. In fact In the absence of all
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perturbation a melted DNA region may be regarded as a freely
jointed chain molecule with the size of Kuhn segment about 7
nucleotidea (L~50 2) /12/. Then for a chain of N segments the
mean distance between the chain ends is d=L"yN/V"2~> /12/
and for a region of ~ 70 bp H=10 and d « 1 1 0 %. If the melted
regions of DNA were not deformed by the electric field, even
very small denatured sections would halt such molecules in gel
electrophoresis. In reality such a melted section in pA03 DBA
only slightly changes its mobility. Presumably the conformation
of a denatured region changes B O drastically in the electric
field (perhaps it is completely stretched out) that DNA molecules with short unwound sections can pass through a 4% polyaorylamide gel. Only a seven times larger melted region
( ^ 5 0 0 bp, d •«# 300 2 in an underformed state) prevents DNA from
entering the gel. A similar value for the size of the melted
region was obtained by Lennan and Pisher / 2 / .
This model accounts for the lack of retardation in ColE1
DNA as opposed to its quarter, pA03 DNA, while both have the
same melted region. The force acting upon a DNA molecule in
an electric field is proportional to the charge, i.e. length,
of the molecule. The resistance offerred by one and the same
melted region does not change, therefore the retardation effect should be stronger in shorter molecules.
In our experiments the melted regions in the DNA molecules
had been fixed with glyoxal which is known to enter a practically irreversible reaction with guanine /13/. To prevent the
unwinding of DNA by glyoxal during fixation a special procedure had been elaborated /4*5/ with the concentration of glyoxal being no higher than 5 all. Because of these precautions we
could not completely prevent renaturation of the fixed region
as a whole /4/. In our fixation procedure the modification of
DNA bases in the melted region distabilized the region and lo=
wered its melting temperature by 20°C (see Figure 6 ) . Therefore until the region is completely melted (55°C) the DNA comprises an array of molecules with unwound regions of different
sizes. Since the retardation effect depends on the size of the
unwound region, the DBA preparation with an incompletely denatured fixed region gives a diffuse band in electrophoresis
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(see Figures 5 ). After the fixed region is completely melted
its size no longer changes. This circumstance was also considered in electron-microscopic studies of DNA melting with the
using the glyoxal procedure /5,6/.
The separation of DNA fragments in a gel with a denaturant
concentration gradient according to Fischer and Lerman /1,2/
involves careful thermostatic control of the gel slabs at 65°C.
In our case the melting temperature of the regions with glyoxal-modified bases is brought down to such an extent that the
addition of the unwinding agents alone suffices for the complete melting of the fixed regions. The long plateau in Figure 6
(~10°C) removes the stringent temperature requirements, so that
electrophoresis can be performed by standard equipment without
special thermostatic control, which makes it a much simpler
procedure.
The retardation effect with glyoxal fixation may also be
applied to denaturation mapping with the use of restriction
maps. Fig. 9 shows that due to fixation of DNA denatured regions In pBR 322 DNA only those electrophoretic bands in DNA
restriction pattern disappear which correspond to the fragments
containing the denatured regions. So If the cleavage map is
known it is possible to localize these fragments on DNA molecule and henoe the regions corresponding to the chosen temperature In the DNA melting range. The use of number of restriction endonuoleases makes for a high acouracy of such mapping
method. A comparison of the electrophoretic patterns of DNA
preparations fixed at different stages of melting in one experiment makes it possible to reconstruct the topography of
DBA melting. This technique may complement the traditional
electron-microscopic method which involves measuring a large
array of molecules in order to construct a melting map. Besides, electron microscopy is usually applied to molecules having a denatured region of more than 100 bp, whereas the retardation effect makeB it possible to detect an unwound region of ~ 70 bp (Figure 4b) even in a molecule ~16OO bp
long.
In conclusion the authors would like to thank Dr. E.P.
Zaichikov, Dr. M.A.Grachev, Dr. A.N.Rekesh, Dr. L.V.Heumyvakin
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and Dr. A.Oka for making a v a i l a b l e the necessary DNA and enzyme p r e p a r a t i o n s and the E . c o l i C6OO s t r a i n with pAO3 p l a s mid, and Prof. Yu.S.Lazurkin, Prof. M.D.Frank-Kamenetskii and
Dr. A.S.Borovik f o r useful comments and s t i m u l a t i n g d i s c u s s i o n s .
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