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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 4813 Nucleic Acids Research 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 4814 Nucleic Acids Research 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. 4815 Nucleic Acids Research 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 ) . 4816 Nucleic Acids Research 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. 4817 Nucleic Acids Research 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 4818 u Nucleic Acids Research 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. 4819 Nucleic Acids Research 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 4820 Nucleic Acids Research 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 4821 Nucleic Acids Research 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. 4822 Nucleic Acids Research 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 4823 Nucleic Acids Research 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 4824 Nucleic Acids Research (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 4825 Nucleic Acids Research 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 . REFERENCES 1. F i s c h e r , S.G. and Lerman, L . S . (1979) C e l l 16, 191-200. 2 . F i s o h e r , S.G. and Lerman, L . S . (1980) Proc.Natl.Acad.Sci. USA 77, 4420-4424. 3 . Mailer, W., H a t t e s o h l , I . , Schuetz, H . - J . and Meyer, G. (1981) tfucl.Acids Res. 9 , 95-119. 4. Pavlov, V.M., Lyubchenko, Yu.L., Borovik, A.S. and Lazurk l n , Yu.S. (1977) Nuol.Acids Res. 4 , 4053-4062. 5. Borovik, A . S . , Kalambet, Yu.A., Lyubchenko, Yu.L. S h i t o v , V.T. and Golovanov, E u . I . (1980) Nucl.Acids Res. 8, 41654184. 6 . P e r e l ' r o i z e n , M.P., Lyamlchev, V . 1 . , Kalambet. 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