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Small angle neutron scattering from DNA molecules during gel electrophoresis
Physica B 180 & 181 (1992) North-Holland
770-772
Small angle neutron electrophoresis
scattering from DNA molecules during gel
F. Cavatorta”, A. Deriua, F. Malizia”, P. Terechb and H.D. Middendorf’ “Dipartimento di Fisica, Vniversitri di Parma, Italy blnstitut Laue-Langevin, Grenoble, France ‘Department of Biochemistry, University of Edinburgh University of Oxford, UK
Medical School and Clarendon Laboratory,
We have performed small angle neutron scattering experiments on agarose-DNA gels undergoing electrophoresis. Two kinds of DNA (5 and 50 kilobase pairs) were used with applied fields up to 5 V/cm. The SANS patterns obtained do not show evidence of any anisotropic scattering. This result is discussed in the context of current theories of DNA fragments migrating through a polysaccharide network.
1. Introduction Gel electrophoresis is a widely used technique to separate macromolecules (DNA and SDS(sodium dodecyl sulphate)-denatured proteins) according to their size. Macromolecules with charged groups reptate through a gel matrix under the driving force of an electric field. The separation obtained is due to the marked dependence of mobility on molecular weight. In both cases, i.e. protein electrophoresis in polyacrylamide gels and DNA electrophoresis using mainly agarose gels, it is important to understand in molecular detail the slow, field-driven, Brownian-like motion of charged biopolymer chains through a semi-rigid, 3D polymer network filled with solvent (i.e. a buffer solution). Since the 196Os, a substantial body of data has accumulated on the effective electrophoretic mobility of biomolecules and their fragments as a function of gel parameters, ionic conditions and field strength [l]. A number of phenomenological relations have been developed and refined to rationalise such data in terms of molecular parameters and quantities characterising the gel structure [2]. Theoretical work on the molecular dynamics of electrophoretic processes has been based mainly on the concept of ‘reptation’ introduced by de Gennes and Edwards [3]. This predicts migration velocities proportional to the applied dc field E and inversely proportional to the mass M of the chain molecule considered. Nonlinear effects come into play at higher M and E, i.e. under conditions which are commonly encountered in DNA electrophoresis (M > 10 kbase), and simulations showed that the mobility as a function of M exhibits a bimodal behaviour [4]. A few semi-quantitative models have been devised to explain this ‘band inversion’ effect [4, 51, but there is yet no molecular-level evidence for or against these 0921-4526/92/$05.00
0
1992 - Elsevier
Science
Publishers
different models. More detailed computer simulations [6, 71 and sophisticated analytical models [8] have been published in recent years. For both fundamental and practical reasons,. neutron techniques are well suited to contribute decisive experimental data to current theoretical work on the molecular interpretation of gel electrophoresis. From an experimental point of view, l-2 mm thick agaroseD,O gel slabs make ideal neutron samples, and steady-state as well as pulsed-field electrophoresis can easily be performed on-beam. Small angle neutron scattering (SANS) experiments, in particular, are able to provide data on the molecular conformation of biopolymer chains during their slow diffusion in the gel matrix. On the basis of the above theoretical models some anisotropy relative to the applied electric field vector E should be detectable in SANS patterns due to the ‘stretching’ of the migrating biopolymer. A prerequisite for such experiments is a good characterisation of ‘empty’ low-concentration gels in terms of interstitial volume size distributions, length and thickness variation of double-helical fibre bundles and extent of higher-order or fractal structure. During the last years, using D17 and Dll, we have studied the large-scale structure of agarose-D,O gels in the lower concentration range relevant to electrophoresis experiments [9, lo]. In the present paper we present results of our first on-beam electrophoresis experiment. 2. Experimental We have used the diffractometer D17 at the ILL to measure small angle scattering from agarose-DNA gels with and without DC electric fields. We employed a specially constructed electrophoresis cell containing 19 x 80 x 2 mm agarose gel samples, with continuous circulation between lower and upper reservoirs to
B.V. All rights
reserved
771
F. Cavatorta et al. I SANS from DNA during gel electrophoresis
permit long run times. Gels were prepared from low melting point agarose in tris phosphate buffer; agarose concentration was 1% throughout. DNA was introduced into the agarose matrix by cogellification; its homogeneity in the beam area was checked by standard staining and UV monitoring techniques. S(Q) data were recorded for two kinds of DNA as follows: (1) 40-50 kbase DNA from calf thymus at a concentration of OS%, at fields E between 0 to 5 V/cm; (2) 2-3 kbase DNA from herring sperm at a concentration of 5%, at fields up to 0.5 V/cm. 3. Results and discussion In order to verify that scattering from DNA was indeed observable at the adopted DNA concentrations, measurements were first performed on pure gels and gels ‘loaded’ with DNA without applied field. As expected, both diffraction patterns are perfectly isotropic. The data were therefore radially averaged and the resulting S(Q) are shown in fig. 1. In the Q-range covered, S(Q) follows a power law of the form S(Q) Q Q -D with an exponent which can be related to both fractality and polydispersity characteristics of the gel [9, lo]. It can be seen that the effect of DNA is to decrease the exponent by about lo%, i.e. from -2.37 to -2.15. However, the SANS patterns obtained for the DNA-loaded gels at different applied fields are also isotropic. As an example, we report in fig. 2 the contour plot pattern for sample 1 at a field of 5 V/cm. Analogous patterns are observed at lower electric fields, and similarly for sample 2. In order to look carefully for small anisotropy effects, we have analysed the intensity profiles Z( Q, 0) in the radial directions defined by the azimuthal angle 19; 0 = 0” (vertical axis) and 90” (horizontal axis) using a Fourier analysis method. In fig. 3 these two intensity profiles are compared, and no significant difference is detected. The same conclusion can be drawn from fig. 4, where experimental data (a) referring to a 0.5% DNA loaded gel with an applied field E = 1.25 V/cm are compared
Fig. 1. Log-log plot of S(Q) for fully D,O-exchanged 1% agarose gel with (0) and without (A) 0.5% DNA (50 kilo base pairs calf thymus).
Fig. 2. Contour intensity plot for (40-50 kilo base pairs calf thymus) vertical applied field E of 5 V/cm.
a 0.5% DNA loaded 1% agarose gel with a
m*~ssqsts*~psIy ’ OO
0.01
0.02
0.03
0.04
0.05
c )6
Q (R’) Fig. 3. Intensity profile 1(Q, 0) in the radial directions defined by the angle 0, obtained by subjecting the 2-dimensional dataset of fig. 2 to a Fourier analysis (up to 32 coefficients in the summation of the sine and cosine terms);
(0) 19= 0” (vertical axis); (A) 8 = 90” (horizontal axis). to the reconstructed image (b) from the above mentioned Fourier analysis. The agreement between the two plots is excellent and confirms that there is no detectable anisotropy. It is difficult to reconcile these results with current ideas about the mode of migration of DNA in gels. Two aspects need to be considered: (i) In the case of relatively short DNA chains, all of them may be disentangled, i.e. ‘sieving’ through the larger gel interstices as blobs with essentially isotropic radii of gyration R,. (ii) In our 40-50 kbase DNA sample, there could be appreciable entanglement with the agarose fibre network, but only over scale lengths ~500 A (due e.g. to high degrees of supercoiling) so that any resulting overall elongation would not be seen in S(Q) patterns with Qmin - 0.011 A-‘. Some authors [ll, 121 have recently questioned the applicability of reptation concepts to DNA gel electrophoresis, and have drawn attention to the wide range
772
F. Cavatorta et al. I SANS from DNA during gel electrophoresis
of scale length also confirmed rose gels.
variations in the gel matrix, which is by our SANS results on ‘empty’ aga-
Acknowledgements We thank Dr. S.E. sample preparation.
Kearsey
for helpful
advise
on
References [II D. Rickwood
PI [31
[41 [51 PI [71 PI
[91 [101 [Ill Fig. 4. Contour intensity plots for a 0.5% DNA loaded (50 kilo base pairs calf thymus) 1% agarose gel (applied electric field E = 1.25 V/cm). (a) Experimental data; (b) Data reconstructed from the Fourier analysis mentioned in the text.
[=I
and B.D. Hames, Gel Electrophoresis of Nucleic Acids (Academic, 1982). S. Waki, J.D. Harvey and A.R. Bellamy, Biopolymers 21 (1982) 1909. R. West, Biopolymers 26 (1987) 111. P.G. de Gennes, Scaling Concepts in Polymer Physics (Cornell Univ., 1979). M. Doianet and S.F. Edwards, The Theory of Polymer Dynamics (Oxford Univ., 1986). G.W. Slater, J. Rousseau and J. Noolandi, Biopolymers 26 (1987) 863. J. Noolani, J. Phys. Rev. Lett. 58 (1987) 2428. A. Baumgartner and M. Muthukumar, Phys. Rev. Lett. 87 (1987) 3082. J. Deutsch, Science 240 (1988) 922. S.F. Edwards and M. Muthukumar, J. Chem. Phys. 89 (1987) 2435. M. Doi et al., Phys. Rev. Lett. 61 (1988) 1893. H.D. Middendorf, F. Cavatorta and A. Deriu, Progr. Coil. Polym. Sci. 81 (1990) 274. A. Deriu, F. Cavatorta, D. Cabrini and H.D. Middendorf, Progr. Coil. Polym. Sci. 84 (1991) 461. B.H. Zimm, Phys. Rev. Lett. 61 (1988) 2965. M. Muthukumar and A. Baumgaertner, Macromolecules 22 (1981) 1937. M. Olvera de la Cruz, D. Gersappe and E.O. Shaffer, Phys. Rev. Lett. 64 (1990) 2324.
770-772
Small angle neutron electrophoresis
scattering from DNA molecules during gel
F. Cavatorta”, A. Deriua, F. Malizia”, P. Terechb and H.D. Middendorf’ “Dipartimento di Fisica, Vniversitri di Parma, Italy blnstitut Laue-Langevin, Grenoble, France ‘Department of Biochemistry, University of Edinburgh University of Oxford, UK
Medical School and Clarendon Laboratory,
We have performed small angle neutron scattering experiments on agarose-DNA gels undergoing electrophoresis. Two kinds of DNA (5 and 50 kilobase pairs) were used with applied fields up to 5 V/cm. The SANS patterns obtained do not show evidence of any anisotropic scattering. This result is discussed in the context of current theories of DNA fragments migrating through a polysaccharide network.
1. Introduction Gel electrophoresis is a widely used technique to separate macromolecules (DNA and SDS(sodium dodecyl sulphate)-denatured proteins) according to their size. Macromolecules with charged groups reptate through a gel matrix under the driving force of an electric field. The separation obtained is due to the marked dependence of mobility on molecular weight. In both cases, i.e. protein electrophoresis in polyacrylamide gels and DNA electrophoresis using mainly agarose gels, it is important to understand in molecular detail the slow, field-driven, Brownian-like motion of charged biopolymer chains through a semi-rigid, 3D polymer network filled with solvent (i.e. a buffer solution). Since the 196Os, a substantial body of data has accumulated on the effective electrophoretic mobility of biomolecules and their fragments as a function of gel parameters, ionic conditions and field strength [l]. A number of phenomenological relations have been developed and refined to rationalise such data in terms of molecular parameters and quantities characterising the gel structure [2]. Theoretical work on the molecular dynamics of electrophoretic processes has been based mainly on the concept of ‘reptation’ introduced by de Gennes and Edwards [3]. This predicts migration velocities proportional to the applied dc field E and inversely proportional to the mass M of the chain molecule considered. Nonlinear effects come into play at higher M and E, i.e. under conditions which are commonly encountered in DNA electrophoresis (M > 10 kbase), and simulations showed that the mobility as a function of M exhibits a bimodal behaviour [4]. A few semi-quantitative models have been devised to explain this ‘band inversion’ effect [4, 51, but there is yet no molecular-level evidence for or against these 0921-4526/92/$05.00
0
1992 - Elsevier
Science
Publishers
different models. More detailed computer simulations [6, 71 and sophisticated analytical models [8] have been published in recent years. For both fundamental and practical reasons,. neutron techniques are well suited to contribute decisive experimental data to current theoretical work on the molecular interpretation of gel electrophoresis. From an experimental point of view, l-2 mm thick agaroseD,O gel slabs make ideal neutron samples, and steady-state as well as pulsed-field electrophoresis can easily be performed on-beam. Small angle neutron scattering (SANS) experiments, in particular, are able to provide data on the molecular conformation of biopolymer chains during their slow diffusion in the gel matrix. On the basis of the above theoretical models some anisotropy relative to the applied electric field vector E should be detectable in SANS patterns due to the ‘stretching’ of the migrating biopolymer. A prerequisite for such experiments is a good characterisation of ‘empty’ low-concentration gels in terms of interstitial volume size distributions, length and thickness variation of double-helical fibre bundles and extent of higher-order or fractal structure. During the last years, using D17 and Dll, we have studied the large-scale structure of agarose-D,O gels in the lower concentration range relevant to electrophoresis experiments [9, lo]. In the present paper we present results of our first on-beam electrophoresis experiment. 2. Experimental We have used the diffractometer D17 at the ILL to measure small angle scattering from agarose-DNA gels with and without DC electric fields. We employed a specially constructed electrophoresis cell containing 19 x 80 x 2 mm agarose gel samples, with continuous circulation between lower and upper reservoirs to
B.V. All rights
reserved
771
F. Cavatorta et al. I SANS from DNA during gel electrophoresis
permit long run times. Gels were prepared from low melting point agarose in tris phosphate buffer; agarose concentration was 1% throughout. DNA was introduced into the agarose matrix by cogellification; its homogeneity in the beam area was checked by standard staining and UV monitoring techniques. S(Q) data were recorded for two kinds of DNA as follows: (1) 40-50 kbase DNA from calf thymus at a concentration of OS%, at fields E between 0 to 5 V/cm; (2) 2-3 kbase DNA from herring sperm at a concentration of 5%, at fields up to 0.5 V/cm. 3. Results and discussion In order to verify that scattering from DNA was indeed observable at the adopted DNA concentrations, measurements were first performed on pure gels and gels ‘loaded’ with DNA without applied field. As expected, both diffraction patterns are perfectly isotropic. The data were therefore radially averaged and the resulting S(Q) are shown in fig. 1. In the Q-range covered, S(Q) follows a power law of the form S(Q) Q Q -D with an exponent which can be related to both fractality and polydispersity characteristics of the gel [9, lo]. It can be seen that the effect of DNA is to decrease the exponent by about lo%, i.e. from -2.37 to -2.15. However, the SANS patterns obtained for the DNA-loaded gels at different applied fields are also isotropic. As an example, we report in fig. 2 the contour plot pattern for sample 1 at a field of 5 V/cm. Analogous patterns are observed at lower electric fields, and similarly for sample 2. In order to look carefully for small anisotropy effects, we have analysed the intensity profiles Z( Q, 0) in the radial directions defined by the azimuthal angle 19; 0 = 0” (vertical axis) and 90” (horizontal axis) using a Fourier analysis method. In fig. 3 these two intensity profiles are compared, and no significant difference is detected. The same conclusion can be drawn from fig. 4, where experimental data (a) referring to a 0.5% DNA loaded gel with an applied field E = 1.25 V/cm are compared
Fig. 1. Log-log plot of S(Q) for fully D,O-exchanged 1% agarose gel with (0) and without (A) 0.5% DNA (50 kilo base pairs calf thymus).
Fig. 2. Contour intensity plot for (40-50 kilo base pairs calf thymus) vertical applied field E of 5 V/cm.
a 0.5% DNA loaded 1% agarose gel with a
m*~ssqsts*~psIy ’ OO
0.01
0.02
0.03
0.04
0.05
c )6
Q (R’) Fig. 3. Intensity profile 1(Q, 0) in the radial directions defined by the angle 0, obtained by subjecting the 2-dimensional dataset of fig. 2 to a Fourier analysis (up to 32 coefficients in the summation of the sine and cosine terms);
(0) 19= 0” (vertical axis); (A) 8 = 90” (horizontal axis). to the reconstructed image (b) from the above mentioned Fourier analysis. The agreement between the two plots is excellent and confirms that there is no detectable anisotropy. It is difficult to reconcile these results with current ideas about the mode of migration of DNA in gels. Two aspects need to be considered: (i) In the case of relatively short DNA chains, all of them may be disentangled, i.e. ‘sieving’ through the larger gel interstices as blobs with essentially isotropic radii of gyration R,. (ii) In our 40-50 kbase DNA sample, there could be appreciable entanglement with the agarose fibre network, but only over scale lengths ~500 A (due e.g. to high degrees of supercoiling) so that any resulting overall elongation would not be seen in S(Q) patterns with Qmin - 0.011 A-‘. Some authors [ll, 121 have recently questioned the applicability of reptation concepts to DNA gel electrophoresis, and have drawn attention to the wide range
772
F. Cavatorta et al. I SANS from DNA during gel electrophoresis
of scale length also confirmed rose gels.
variations in the gel matrix, which is by our SANS results on ‘empty’ aga-
Acknowledgements We thank Dr. S.E. sample preparation.
Kearsey
for helpful
advise
on
References [II D. Rickwood
PI [31
[41 [51 PI [71 PI
[91 [101 [Ill Fig. 4. Contour intensity plots for a 0.5% DNA loaded (50 kilo base pairs calf thymus) 1% agarose gel (applied electric field E = 1.25 V/cm). (a) Experimental data; (b) Data reconstructed from the Fourier analysis mentioned in the text.
[=I
and B.D. Hames, Gel Electrophoresis of Nucleic Acids (Academic, 1982). S. Waki, J.D. Harvey and A.R. Bellamy, Biopolymers 21 (1982) 1909. R. West, Biopolymers 26 (1987) 111. P.G. de Gennes, Scaling Concepts in Polymer Physics (Cornell Univ., 1979). M. Doianet and S.F. Edwards, The Theory of Polymer Dynamics (Oxford Univ., 1986). G.W. Slater, J. Rousseau and J. Noolandi, Biopolymers 26 (1987) 863. J. Noolani, J. Phys. Rev. Lett. 58 (1987) 2428. A. Baumgartner and M. Muthukumar, Phys. Rev. Lett. 87 (1987) 3082. J. Deutsch, Science 240 (1988) 922. S.F. Edwards and M. Muthukumar, J. Chem. Phys. 89 (1987) 2435. M. Doi et al., Phys. Rev. Lett. 61 (1988) 1893. H.D. Middendorf, F. Cavatorta and A. Deriu, Progr. Coil. Polym. Sci. 81 (1990) 274. A. Deriu, F. Cavatorta, D. Cabrini and H.D. Middendorf, Progr. Coil. Polym. Sci. 84 (1991) 461. B.H. Zimm, Phys. Rev. Lett. 61 (1988) 2965. M. Muthukumar and A. Baumgaertner, Macromolecules 22 (1981) 1937. M. Olvera de la Cruz, D. Gersappe and E.O. Shaffer, Phys. Rev. Lett. 64 (1990) 2324.