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Transverse agarose pore gradient gel electrophoresis of DNA
Journal of Biochemical and Biophysical Methods, 24 (1992) 181-194
181
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00932
Transverse agarose pore gradient gel electrophoresis of DNA John S. Fawcett, David Wheeler and Andreas Chrambach Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA (Received 16 September 1991) (Revision received 25 November 1991) ( ~ c e p t e d 26 November 1991)
Summary Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a l-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180). Key words: Electrophoresis, agarose gradient; Agarose gradient, transverse; Ferguson curve, computer traced; Electrophoresis, semi-automated; DNA; DNA, conformation
Introduction The joint report dealing with a new semi-automated method for Ferguson plot analysis by transverse polyacrylamide pore gradient gel electrophoresis [1] has summarized the reasons for doing Ferguson plot analysis, and for using transverse Abbreviations: Ferguson curve, migration distance vs. gel concentration; Ferguson plot, log(mobility) vs. gel concentration; K R, retardation coefficient, slope of the Ferguson curves plotted semilogarithmically (Ferguson plot); SPADNS, 2-(p-sulfophenylazo)-l,8-dihydroxy-3,6-naphthalene disulfonic acid, trisodium salt; %T, total gel concentration; Yo, intercept of the Ferguson plot with the mobility axis at %T = 0. Correspondence address: A. Chrambach, Bldg. 10, Rm. 6C101, NIH, Bethesda MD 20892, USA.
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pore gradient gels for that purpose. In an attempt to extend that method of electrophoretic analysis to larger sizes of DNA than amenable to polyacrylamide gel electrophoresis, i.e. to sizes beyond 10 kb [2], this study aimed at a method for forming transverse pore gradient gels in agarose. The development of that method was aided by the previous elaboration of a method for producing conventionally oriented agarose pore gradient gels [3].
Materials and Methods
Agarose was SeaKem GTG (FMC, Rockland, ME). Gel buffer, catholyte and anolyte were 0.5 x TBE (0.045 M Tris, 0.045 M boric acid, 0.001 M NazEDTA , pH 8.4). GelBond was obtained from FMC (Cat. No. 53761). SPADNS (2-(psulfophenylazo)-l,8-dihydroxy-3,6-naphthalene disulfonic acid, trisodium salt)was obtained from Aldrich (Milwaukee, WI). Silicone oil DC 200 (10 cst)was a product of Serva Fine Biochemica (Heidelberg, Germany). DNA was a 1-kb ladder (BRL, Life Techn., Gaithersburg, MD, Cat. No. 5615SB)with 17 component fragments between 298 and 12 216 bp in length. Prior to electrophoresis, some DNA samples were fluorophore labeled, using ethidium homodimer (Molecular Probes, Eugene, OR, Cat. No. E-1169) [4]. Preparation of the agarose pore gradient gel The cassette for forming the pore gradient gel (Fig. 1) consisted of 6 layers (in the case of photography of the cassette under reflective illumination) or 4 layers (in the case of cassette photography with transmitted light) oriented from front to back as follows:
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Fig. 1. Cassette for the formation of transverse agarose pore gradient gels. (A) Loxan plate with bottom hole; (B) Tygon tubing sealed into the hole of (A); (C) sample slot former; (D) rubber gasket; (E) GelBond; (F) glass plate; (G) white card board; (H) glass or aluminum back plate.
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• Lexan plate A (125 × 220 x 8 mm) with I hole, with Tygon tubing B sealed-in by silicone nlbber adhesive (RTV, General Electric, Waterford, NY), at a central position 2 cm from the bottom of the plate. A sample slot former (2 × 1 × 130 mm) C is epoxy-glued to the plate at 28 mm from the edge of the plate and 55 mm from its bottom. • Rubber gasket D (3 mm thick and 14 mm wide, V-shaped at the bottom). • GelBond (124 × 220 × 0.2 mm) E which, with its hydrophobic side facing downward, was rolled [5] onto glass plate F on which a few drops of silicone oil (viscosity 10 cs0 were spread to eliminate entrapped air bubbles. • Glass plate F (4 mm thick, other dimensions as A). • White cardboard G (125 × 220 mm). • Glass or aluminum back plate H (dimensions as A). Items G and H were omitted when the gradient was analyzed under transillumination. The light solution was 0.2% agarose in buffer (50 ml). The heavy solution was 1.5% agarose in buffer containing 20% glycerol and SPADNS or bromphenolblue dye (50 ml). Agarose solutions were prepared by heating to the boiling point in a microwave oven as described [6]. The cassette was filled from the bottom by upward displacement of the density gradient. This involves adding heavy solution to the light solution in the gradient former. To facilitate mixing, it is therefore preferable to add the heavy solution to the top of the solution in the mixing syringe (i.e. via the plunger). The gradient maker previously described [7] was therefore modified by changing the position of the entrance port of the heavy solution into the light one from the bottom of syringe A to an entrance through the plunger of 20-
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Fig. 2. Diagramatic representation of the agarose pore gradient gel maker. The previous design of the syringe holder of the gradient maker [7] was modified as shown since the cassette was filled from the bottom, therefore syringe B contained the more dense ('heavy') solution, and mixing was improved when the heavy solution entered syringe A via the plunger. (A, B) 50 ml plastic syringes; (A~, B~) syringe plungers; (A a, B 3) connecting tubes; (A 3) outlet tube; (A4) magnetic stirring bar; (C) interchangeable syringe holder plate; (D) mobile platform for plunger depression; (G) spacer bar of adjustable height; the bar is removed prior to displacement of the syringes.
184
syringe A (Fig. 2). The line carrying the heavy solution was sealed into the flexible tip portion of the syringe plunger by RTV adhesive. The syringe holder was positioned in a thermostated waterbath maintained at 65°C and resting on top of a magnetic stirrer (part H of Ref. 7). The spacer (part G) was 7.5 cm high in application to the cassette described below. Syringe B was connected to a 30 cm length of Tygon tubing (1/32" ID, Thomas Scientific, Swedesboro, NJ, Cat. No. 2848-H22) closed at the end by a clamp. Syringe A was connected to 8 cm of the same tubing and clamped at the bottom; the tubing leading through the plunger was 25 cm in length. Syringe A was filled with light solution (35 ml or more), syringe B with heavy solution (35 ml or more) immediately after microwaving. Syringe B after filling was inverted, the clamp opened and the plunger depressed to displace all air from the syringe and tube which was then clamped. Syringe A containing the stirring bar was filled through tubing A2 leading through the plunger with the bottom tubing clamped. Tube A2 was then clamped, the syringe was inverted, bottom tube A3 was unclamped and the plunger was depressed to displace all air from syringe and tubing A3 which was then clamped. Syringes A and B were then inserted into the syringe holder, tubes A3 and B3 were unclamped, spacer G was inserted between plates C and D, and D was depressed until it made contact with spacer G. A3 was clamped, A2 was unclamped and connected to B3 via a silicone rubber sleeve (3/16" ID). A3 was unclamped and connected to inlet tube B (Fig. 1) via a silicone rubber sleeve (as above). The syringe assembly was then immersed into the thermostated water bath located inside of a closed heated cabinet maintained at 65°C and with a front glass door and an air circulating fan. The solutions within the 2 syringes and the cassette were allowed to equilibrate to 65°C for 1 h. The magnetic stirrer was activated during that time. During that hour of equilibration, the baseline for acquisition of the gradient (see next section) can be obtained by photography of the empty cassette. The motor-driven syringe was activated (part O of Ref. 7). The syringes were emptied within 14 min (plunger displacement rate 0.4 cm/min regulated by variable speed control N (7). Nominally, the agarose concentration range of the gradient was 0.3 to 1.3 (Fig. 3A) and 0.2 to 1.5% (Fig. 3B). In reality, due to the design of the gradient making apparatus and the volume of the solution in the connecting tube, the gel concentration range is narrowed. The gradient in the cassette starts after an initial volume, 1I, of the light solution (V= VA~+ 2 VA, (subscripts refer to part numbers in Fig. 2)) has entered the cassette. The slope of the gradient is therefore derived from a total volume of 2 × (initial VA - VA,). In calculating the initial volume, VA, allowance is made for the volume of the stirring bar. However, the usable volume of the gradient is reduced by the volume in the V-shaped bottom segment of the cassette (darkened in Fig. 2) and the volumes in the connecting tubes, as well as the volume surrounding the magnetic stirrer in syringe A and the volume remaining in syringe B. Furthermore, the sample slot does not extend to the full width of the gel, and for the setup described these corrections reduce the nominal concentration limits of the agarose gradient of 0.3-1.3% to 0.33-1.08%, and the 0.2-1.5% to 0.23-1.21%.
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Documentation of the gradient, using the dye incorporated into the gelation mixture as a measure of agarose concentration The agarose concentration gradient was evaluated in 2 ways: by photography followed by video acquisition and by densitometry of the gel. The cassette was directly illuminated within the cabinet by a fluorescent light tube (60 W) positioned at an angle to the glass door of the cabinet, above and behind the camera, so as to minimize reflections. Photography of the cassette was conducted by Polaroid camera as described previously [6]. Photographic negatives were video-photographed and transformed to computer images by use of program IMAGE (version 1.32) of W.D. Rasband [8], using a Macintosh II personal computer. The images were evaluated to yield grey-level values directly related to dye concentrations. Standards were prepared as a series of concentrations of the dye in agarose formed by mixing hot heavy and light solutions in appropriate proportions prior to gelation. Transverse agarose gradient gels and 5 single concentration agarose standard gels incorporating SPADNS and supported by GelBond were subjected to densitometry on the Biomed densitometer (Model 504, calibrated and tested for accuracy [9]). Correction for the standards was made on the basis of a 2nd-order polynomial function derived from the 5 standards relating optical density and agarose concentration. Agarose gel electrophoresis of DNA Clamps were removed from the cassette. Plate A (Fig. 1) was lifted, starting at the bottom of the cassette containing the high-concentration gel, separating it from the rubber gasket by spatula. Glass plate H, cardboard G and glass plate F were separated from GelBond E, and the gasket was pulled off the GelBond. The bottom triangular section of the gel attached to GelBond was cut off by scissors. The top of the GelBond was trimmed to the width of the ele~trophoresis apparatus (155 mm in application to a BioRad DNA Sub Cell apparatus used). The gel was washed to free it of dye and glycerol either by 3 changes of I I buffer over 48 h, or by 2 changes of 1 1 water followed by 2 changes of buffer over the same period. The washed gel on its GeIBond support was placed into the electrophoresis apparatus and covered with buffer to a height of 2 mm. DNA sample was prepared by diluting 2/~1 of the solution of the 1-kb ladder (1 mg/ml) stored at 4°C with 60 /~1 0.25 x TBE buffer containing 10% glycerol and SPADNS. In some separations (e.g. Fig. 3A), DNA was prelabeled with ethidium homodimer as described [4]. The entire volume was• underlayered into the sample slot using an Eppendorf syringe with Multiflex ~dps. Electrophoresis was conducted at a regulated voltage across the apparatus of 50 V (25 V per 9.8 cm of gel) until the dye had just migrated off the gel. In separations (e.g. Fig. 3B) where DNA had not been prelabeled, the gel with its support was transferred into a tray containing 200 ml of 0.5 /~g/ml ethidium bromide and allowed to stain for 30 min. The wet gel was placed with the GelBond facing upward onto a UV-transilluminator (Fotodyne, New Berlin, WI) the surface of which was covered with SaranWrap to prevent contamination. The
187
gel was photographed using a 2 min exposure and the negative was fixed as previously described [6].
Data processing The photographic negative was viewed by a CCD video camera interfaced with a personal computer as described (see Image acquisition by the computer [1]). Through the interface of that system the Ferguson curves were acquired and digitized. The digitized Ferguson curves were plotted and subjected to least-square linear regression analysis to give values of slope and intercept with the mobility axis at %T = 0 [1].
Results
Transverse agarose pore gradient gel pattern of DNA Transverse agarose pore gradient gel electrophoresis (nominal 0.3-1.3% (Fig. 3A) or 0.2-1.5% (Fig. 3B) SeaKem GTG, 0.5 x TBE buffer, pH 8.4, 20°C) of fluorophore-labeled DNA (1-kb ladder) is shown in Fig. 3. Bromphenol blue (Fig. 3A) or SPADNS (Fig. 3B) was added to the sample and migrated ahead of the DNA zones (the dye was run off just prior to termination of the electrophoretic 0.5% Agarose
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Fig. 5. Pore gradient analysis based on photography of the dye gradient prior to gelation followed by digitization. The gradient mixture by which the gel shown in Fig. 3A was formed was photographed and the negative was video-photographed. Video pictures were transformed to computer images and digitized as described under Materials and Methods. The digitized concentration profile of the hot gradient mixture is shown. Low grey levels correspond to high agarose concentrations.
separations shown in the figure). Representative Ferguson curves were correlated with DNA length (bp) as described in Materials and Methods. As a control experiment, a constant 0.5% agarose gel incorporating a 0-20% glycerol gradient was prepared and washed under identical conditions as used for the agarose gradient gels. The 1-kb DNA ladder migrated in this gel as zones parallel to the starting slot (Fig. 4) showing that the glycerol was quantitatively washed out.
Densitornetric gradient profiles of incorporated dye in the hot gradient mixture and in the agarose gel The photographic negative of the cassette containing the hot agarose gradient
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Fig. 6. Densitometric tracing of a transverse agarose pore gradient gel. The gel formed from the gradient mixture shown in Fig. 3B by overnight gelation at a temperature gradually decreasing to room temperature was scanned in a Biomed densitometer to yield die concentration profile of SP,SJ~NS shown in the figure (uncorrected). The correction was derived from codensitometry of a range of 5 single concentration agarose gels incorporating SPADNS. The density values were fitted with a 2nd-order polynomial function (inset figure) for purposes of interpolation.
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containing a superimposed glycerol-stabilized Bromphenol blue gradient was used for color density acquisition by video camera. The image was digitized and stored in a personal computer. The digitized image was used to compute the agarose concentration in the hot gradient solution (Fig. 5). A conventional densitometry pattern of a representative agarose pore gradient gel of the composition of the one shown in Fig. 3B after overnight gelation through gradual cooling to room temperature is shown in Fig. 6. The densitometric scan was corrected by use of agarose concentration standards incorporating SPADNS to give a scan of increased linearity.
Evaluation of the Ferguson curves formed in agarose gels The Ferguson curves shown in Fig. 3 were acquired in a personal computer as described under Materials and Methods. The digitized curves shown in Fig. 7 were evaluated by least-square linear regression analysis, yielding the slopes, K R, and intercepts on the mobility axis at %T = 0, Yo, depicted in Fig. 8. DNA length (bp) was derived on the basis of a comparison, regarding the relative spacing and intensity of bands, with the gel pattern shown by the supplier. Curves 8A and 8B derived from independent experiments and different concentration ranges of the agarose gradient appear similar, in spite of the difference in labeling.
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Discussion
What is the importance of the transverse agarose pore gradient gel for DNA electropho~esis? The joint report on transverse pore gradient gels formed in polyacrylamide has given a detailed rationale for transverse pore gradient gel electrophoresis in general [1]. In essence, the importance of these gels derives from that of Ferguson plot analysis (reviewed in [10]), which uses electrophoretic mobility values derived
191
from gels of various concentrations to gain insight into molecular conformation and free electrophoretic mobility. As more and more cases of non-linear Ferguson plots become known, and as multi-component DNA systems gain more and more importance, the Ferguson plot analysis required for that purpose becomes excessively laborious and, in case of multicomponent systems, unfeasible without assuming uniformity of net charge density and conformation among all of the components. The transverse pore gradient gel avoids these problems by providing a continuous 'Ferguson curve' over the concentration range of the gradient. In application to DNA, polyacrylamide gels are limited to the DNA length range up to approx. 15 kb; for larger DNA, up to a size of approximately 50 kb, electrophoresis in agarose gels is used [2,11]. Therefore, to potentially secure the benefits of Ferguson plot analysis based on 'Ferguson curves' for the larger size class of DNA, a method of preparing transverse agarose pore gradient gels was developed. Qualitatively, the information with regard to size and conformation (KR) and to free mobility (Yo) from transverse agarose gradient gels corresponds to that described for polyacrylamide in the joint report [1]. However, Ferguson curves are more linear (compare Fig. 3 with Fig. 1 of [1]). Correspondingly, the plot of K R vs. DNA length is convex (Fig. 8) while it is concave when derived from transverse polyacrylamide pore gradient gels (Fig. 3A of [1]). The plots of Yo vs. DNA lengths (Fig. 3B of [1] and Fig. 8) appear indistinguishable, although the paucity of agarose data in the 5 to 10-kb range of DNA precludes a verification in that medium of the concave curvature of the plot. The determination of gel concentration at each point of a transverse pore gradient gel Just as the laboriousness of individual gel formation at many gel concentrations and the uncertainty of relating band positions between those gels is the Achilles' heel of the original Fergu~fon plot method, the pore gradient gel is burdened by the need to ascertain accurately the gel concentration at each point along the pore gradient. Fast-forming polyacrylamide pore gradient gels can be analyzed by densitometry of a dye incorporated into the gradient with the reasonable expectation that dye diffusion is negligible within the short polymerization time, and in gels of relatively high and restrictive gel concentrations [9]. The diffusion of the dye gradient has also been previously neglected in application to agarose gels subjected to densitometry immediately after gelation [3]. However, such an analysis is not optimal since uniformly and fully gelled agarose gels require slow cooling over an extended gelation time, usually carried out overnight. Therefore, an attempt has been made in this study to evaluate the dye gradient prior to gelation, i.e. immediately after gradient formation. This evaluation involved relatively costly instrumentation, viz. a video camera and computer interface for the sake of convenience. More simple photographic and densitometric apparatus could have been used to avoid the error in gel concentration assignment due to overnight dye diffusion. To date, however, this approach to quantitation of %T, using photography of the hot agarose solution prior to gelation of the gradient followed by video acquisition and digitization, has failed as yet to yield an accurate quantitation due
192
to our inability with the available setup to eliminate light reflections from the glass surfaces surrounding the cassette; illumination from the back of the cassette also failed. Fig. 5 illustrates the method of documentation of the gradient mixture by photography, video acquisition and digitization, although it was carried out with non-uniform illumination of the cassette. The sigmoidal shape of the gradient resembles that found previously [3]. With the gradient former described, although a linear concentration gradient is theoretically possible, it would in practice only be obtained if the mixing in the syringe was complete before any other solution was displaced in the cassette, i.e. it operated at an infinitely slow flow rate. In reality, complete mixing of the heavy solution into the light solution is never achieved before some of the light solution has been displaced from the syringe, resulting initially in a slightly lower concentration than predicted. As the gradient proceeds, there will be an excess of heavy solution in the mixing syringe, giving rise to a higher concentration than predicted for the latter half of the gradient. Both photography (or video photography) of the gelation mixture and of the gel do not provide a linear response to concentration of the dye and therefore would require internal standards; an array of 7 dye concentration standards in the range of interest can be displayed above the cassette and co-photographed for that purpose, but to date, it remains inaccurate in application to the gradient mixture prior to gelation due to failure to provide a uniform illumination of the cassette (see above). A corresponding correction for the non-linear densitometric response to color intensity is exemplified in Fig. 6. The analysis of the concentration gradient by the optical density of the dye gradient as used in this study gives a continuous profile and appears preferable to gradient analysis by the discontinuous slicing and weighing of the gel (section 2.5 of [12]).
Justification for gel washing While agarose gel gradients over a wide concentration range can be density stabilized without additives [13], pore gradient gels in the range of 0.3 to 1.3% agarose require the addition of a dense medium to the heavy solution, such as the heat-stable Nycodenz [9] or D20 [3]. For making single agarose pore gradient gels the previously used method of density stabilization of the gelation mixture by D20 [3] is preferable to that by glycerol used in the present study since D20 does not increase the viscosity of the gel and therefore does not have to be re/noved prior to electrophoresis. Glycerol was used here, and the additional washing step burdening the procedure was accepted, in view of the long-term goal of preparing batches of multiple pore gradient gels by a single gelation. For a batchwise preparation of these pore gradient gels, it would be possible to monitor on a single representative gel of each batch the gel concentrations within the gradient by dye incorporation into the gelation mixture as in this study, without being forced to conduct the electrophoresis in gels containing the dye, as long as the dye is washed out of the gels prior to electrophoresis. Since for that reason gel washing is desirable, substitution of the costly density stabilizing medium, D20 , by inexpensive glycerol imrJoses no additional burdens on the procedure. The effectiveness of glycerol removal from the agarose gradient gel was demon-
193
strated by showing that zones of DNA electrophoresed at a right angle to the orientation of the glycerol gradient were uniformly straight (Fig. 4). More importantly yet, the apparent independence of the migration rate and therefore, effective pore size, on the amount of glycerol present during gelation rules out, at least for the 0.5% gel, a dependence of effective pore size on glycerol within the concentration range used.
Horizontally vs. vertically oriented transverse pore gradient gels The horizontal mode of transverse pore gradient gel electrophoresis has two advantages over the vertical mode described in the joint report [1]: the sample slot replacing the gel surface as the starting zone avoids the necessary unevenness of the latter which is due to the fact that after gelation, spacers need to be removed from it. This unevenness of the starting zone in the vertical orientation perturbs the gel pattern. The second advantage of the horizontal orientation is its compatibility with the widely practiced submarine mode of DNA electrophoresis. Limitations of the procedure In the present study, the required accuracy of the method in defining the agarose concentration has not been achieved and further work is necessary to refine this approach, especially in obtaining uniform illumination conditions for the in situ video photography of the cassette. Furthermore, to become a practical tool for Ferguson plot analysis of DNA, transverse pore gradient gels will have to be produced batchwise. The unit gel of such a batch would be nearly identical to that presented in this study; however, the much increased volume of gel would require a gradient maker with larger volume capacity than the one presently used [7]. Since the precision of gel concentration values within the gradient depends on the precision of gradient formation, a computer-directed gradient maker [14] should be the instrument of choice for batchwise preparation of agarose pore gradient gels.
Simplified description of the method The formation of pore gradient agarose gels oriented at a right angle to the direction of electrophoresis (transverse agarose pore gradient gels) is described. Each component on these gels describes a curve which corresponds to a plot of migration distance vs. gel concentration. The information regarding size and surface net charge of the component and regarding properties of the gel fiber inherent in those curves is evaluated by video acquisition of the gel pattern and its digitization in a personal computer. One important application of the method is in the detection of DNA species and complexes with abnormal conformations and/or free electrophoretic mobilities.
References 1 Wheeler, D., Orban, L., Garner, M.M. and Chrambach, A. (1992) J. Biochem. Biophys. Methods, 24, 171-180.
194 2 SteUwagen, N.C. (1987) In: Chrambach, A., Dunn, M.J. and Radola, B.J., (Eds.), Advances in Electrophoresis Vol. 1, pp. 177-228, VCH, Weinheim, Germany. 3 Gombocz, E. and Chrambach, A. (1991) Electrophoresis 12 (12), (in press). 4 Glazer, A.N., Peck, K. and Mathies, R.A. (1990) Proc. Natl. Acad. Sci. USA 87, 3851-3855. 5 Application Note 324, LKB, Bromma, Sweden. 6 Gombocz, E., Tietz, D., Hurtt, S,S. and Chrambach, A. (1987) Electrophoresis 8, 261-271. 7 Fawcett, J.S., Sullivan, J.V., Chidakel, B.E. and Chrambach, A. (1988) Electrophoresis 9, 216-220. 8 Hook, G.R. and Rasband, W. (1987) In: Bailey, G.W. (Ed.), Proc. 45th Ann. Meet. Electron Microsc. Soc. Am. pp. 920-921, San Francisco Press, San Francisco CA. 9 Tietz, D., Gombocz, E. and Chrambach, A. (1991) Electrophoresis 12, 712-721. 10 Tietz, D. (1988) In: Chrambach, A., Dunn, M.J. and Radola, B.J. (Eds.), Advances in Electrophoresis Vol. 2, pp. 109-170, VCH, Weinheim, Germany.. 11 Orban, L. and Chrambach, A. (1991) Electrophoresis 12, 233-240. 12 Fawcett, J.S., Sullivan, J.V. and Chrambach, A. (1989) Electrophoresis 10, 182-185. 13 Coulson, S.E. and Cook, R.B. (1983) In: Stathakos, D. (Ed.), Electrophoresis '82, pp. 363-370, W. de Gruyter, Berlin, Germany. 14 Altland, K. and Altland, A. (1984) Electrophoresis 5, 143-147.
181
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-022X/92/$05.00
JBBM 00932
Transverse agarose pore gradient gel electrophoresis of DNA John S. Fawcett, David Wheeler and Andreas Chrambach Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA (Received 16 September 1991) (Revision received 25 November 1991) ( ~ c e p t e d 26 November 1991)
Summary Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a l-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180). Key words: Electrophoresis, agarose gradient; Agarose gradient, transverse; Ferguson curve, computer traced; Electrophoresis, semi-automated; DNA; DNA, conformation
Introduction The joint report dealing with a new semi-automated method for Ferguson plot analysis by transverse polyacrylamide pore gradient gel electrophoresis [1] has summarized the reasons for doing Ferguson plot analysis, and for using transverse Abbreviations: Ferguson curve, migration distance vs. gel concentration; Ferguson plot, log(mobility) vs. gel concentration; K R, retardation coefficient, slope of the Ferguson curves plotted semilogarithmically (Ferguson plot); SPADNS, 2-(p-sulfophenylazo)-l,8-dihydroxy-3,6-naphthalene disulfonic acid, trisodium salt; %T, total gel concentration; Yo, intercept of the Ferguson plot with the mobility axis at %T = 0. Correspondence address: A. Chrambach, Bldg. 10, Rm. 6C101, NIH, Bethesda MD 20892, USA.
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pore gradient gels for that purpose. In an attempt to extend that method of electrophoretic analysis to larger sizes of DNA than amenable to polyacrylamide gel electrophoresis, i.e. to sizes beyond 10 kb [2], this study aimed at a method for forming transverse pore gradient gels in agarose. The development of that method was aided by the previous elaboration of a method for producing conventionally oriented agarose pore gradient gels [3].
Materials and Methods
Agarose was SeaKem GTG (FMC, Rockland, ME). Gel buffer, catholyte and anolyte were 0.5 x TBE (0.045 M Tris, 0.045 M boric acid, 0.001 M NazEDTA , pH 8.4). GelBond was obtained from FMC (Cat. No. 53761). SPADNS (2-(psulfophenylazo)-l,8-dihydroxy-3,6-naphthalene disulfonic acid, trisodium salt)was obtained from Aldrich (Milwaukee, WI). Silicone oil DC 200 (10 cst)was a product of Serva Fine Biochemica (Heidelberg, Germany). DNA was a 1-kb ladder (BRL, Life Techn., Gaithersburg, MD, Cat. No. 5615SB)with 17 component fragments between 298 and 12 216 bp in length. Prior to electrophoresis, some DNA samples were fluorophore labeled, using ethidium homodimer (Molecular Probes, Eugene, OR, Cat. No. E-1169) [4]. Preparation of the agarose pore gradient gel The cassette for forming the pore gradient gel (Fig. 1) consisted of 6 layers (in the case of photography of the cassette under reflective illumination) or 4 layers (in the case of cassette photography with transmitted light) oriented from front to back as follows:
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Fig. 1. Cassette for the formation of transverse agarose pore gradient gels. (A) Loxan plate with bottom hole; (B) Tygon tubing sealed into the hole of (A); (C) sample slot former; (D) rubber gasket; (E) GelBond; (F) glass plate; (G) white card board; (H) glass or aluminum back plate.
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• Lexan plate A (125 × 220 x 8 mm) with I hole, with Tygon tubing B sealed-in by silicone nlbber adhesive (RTV, General Electric, Waterford, NY), at a central position 2 cm from the bottom of the plate. A sample slot former (2 × 1 × 130 mm) C is epoxy-glued to the plate at 28 mm from the edge of the plate and 55 mm from its bottom. • Rubber gasket D (3 mm thick and 14 mm wide, V-shaped at the bottom). • GelBond (124 × 220 × 0.2 mm) E which, with its hydrophobic side facing downward, was rolled [5] onto glass plate F on which a few drops of silicone oil (viscosity 10 cs0 were spread to eliminate entrapped air bubbles. • Glass plate F (4 mm thick, other dimensions as A). • White cardboard G (125 × 220 mm). • Glass or aluminum back plate H (dimensions as A). Items G and H were omitted when the gradient was analyzed under transillumination. The light solution was 0.2% agarose in buffer (50 ml). The heavy solution was 1.5% agarose in buffer containing 20% glycerol and SPADNS or bromphenolblue dye (50 ml). Agarose solutions were prepared by heating to the boiling point in a microwave oven as described [6]. The cassette was filled from the bottom by upward displacement of the density gradient. This involves adding heavy solution to the light solution in the gradient former. To facilitate mixing, it is therefore preferable to add the heavy solution to the top of the solution in the mixing syringe (i.e. via the plunger). The gradient maker previously described [7] was therefore modified by changing the position of the entrance port of the heavy solution into the light one from the bottom of syringe A to an entrance through the plunger of 20-
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Fig. 2. Diagramatic representation of the agarose pore gradient gel maker. The previous design of the syringe holder of the gradient maker [7] was modified as shown since the cassette was filled from the bottom, therefore syringe B contained the more dense ('heavy') solution, and mixing was improved when the heavy solution entered syringe A via the plunger. (A, B) 50 ml plastic syringes; (A~, B~) syringe plungers; (A a, B 3) connecting tubes; (A 3) outlet tube; (A4) magnetic stirring bar; (C) interchangeable syringe holder plate; (D) mobile platform for plunger depression; (G) spacer bar of adjustable height; the bar is removed prior to displacement of the syringes.
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syringe A (Fig. 2). The line carrying the heavy solution was sealed into the flexible tip portion of the syringe plunger by RTV adhesive. The syringe holder was positioned in a thermostated waterbath maintained at 65°C and resting on top of a magnetic stirrer (part H of Ref. 7). The spacer (part G) was 7.5 cm high in application to the cassette described below. Syringe B was connected to a 30 cm length of Tygon tubing (1/32" ID, Thomas Scientific, Swedesboro, NJ, Cat. No. 2848-H22) closed at the end by a clamp. Syringe A was connected to 8 cm of the same tubing and clamped at the bottom; the tubing leading through the plunger was 25 cm in length. Syringe A was filled with light solution (35 ml or more), syringe B with heavy solution (35 ml or more) immediately after microwaving. Syringe B after filling was inverted, the clamp opened and the plunger depressed to displace all air from the syringe and tube which was then clamped. Syringe A containing the stirring bar was filled through tubing A2 leading through the plunger with the bottom tubing clamped. Tube A2 was then clamped, the syringe was inverted, bottom tube A3 was unclamped and the plunger was depressed to displace all air from syringe and tubing A3 which was then clamped. Syringes A and B were then inserted into the syringe holder, tubes A3 and B3 were unclamped, spacer G was inserted between plates C and D, and D was depressed until it made contact with spacer G. A3 was clamped, A2 was unclamped and connected to B3 via a silicone rubber sleeve (3/16" ID). A3 was unclamped and connected to inlet tube B (Fig. 1) via a silicone rubber sleeve (as above). The syringe assembly was then immersed into the thermostated water bath located inside of a closed heated cabinet maintained at 65°C and with a front glass door and an air circulating fan. The solutions within the 2 syringes and the cassette were allowed to equilibrate to 65°C for 1 h. The magnetic stirrer was activated during that time. During that hour of equilibration, the baseline for acquisition of the gradient (see next section) can be obtained by photography of the empty cassette. The motor-driven syringe was activated (part O of Ref. 7). The syringes were emptied within 14 min (plunger displacement rate 0.4 cm/min regulated by variable speed control N (7). Nominally, the agarose concentration range of the gradient was 0.3 to 1.3 (Fig. 3A) and 0.2 to 1.5% (Fig. 3B). In reality, due to the design of the gradient making apparatus and the volume of the solution in the connecting tube, the gel concentration range is narrowed. The gradient in the cassette starts after an initial volume, 1I, of the light solution (V= VA~+ 2 VA, (subscripts refer to part numbers in Fig. 2)) has entered the cassette. The slope of the gradient is therefore derived from a total volume of 2 × (initial VA - VA,). In calculating the initial volume, VA, allowance is made for the volume of the stirring bar. However, the usable volume of the gradient is reduced by the volume in the V-shaped bottom segment of the cassette (darkened in Fig. 2) and the volumes in the connecting tubes, as well as the volume surrounding the magnetic stirrer in syringe A and the volume remaining in syringe B. Furthermore, the sample slot does not extend to the full width of the gel, and for the setup described these corrections reduce the nominal concentration limits of the agarose gradient of 0.3-1.3% to 0.33-1.08%, and the 0.2-1.5% to 0.23-1.21%.
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Documentation of the gradient, using the dye incorporated into the gelation mixture as a measure of agarose concentration The agarose concentration gradient was evaluated in 2 ways: by photography followed by video acquisition and by densitometry of the gel. The cassette was directly illuminated within the cabinet by a fluorescent light tube (60 W) positioned at an angle to the glass door of the cabinet, above and behind the camera, so as to minimize reflections. Photography of the cassette was conducted by Polaroid camera as described previously [6]. Photographic negatives were video-photographed and transformed to computer images by use of program IMAGE (version 1.32) of W.D. Rasband [8], using a Macintosh II personal computer. The images were evaluated to yield grey-level values directly related to dye concentrations. Standards were prepared as a series of concentrations of the dye in agarose formed by mixing hot heavy and light solutions in appropriate proportions prior to gelation. Transverse agarose gradient gels and 5 single concentration agarose standard gels incorporating SPADNS and supported by GelBond were subjected to densitometry on the Biomed densitometer (Model 504, calibrated and tested for accuracy [9]). Correction for the standards was made on the basis of a 2nd-order polynomial function derived from the 5 standards relating optical density and agarose concentration. Agarose gel electrophoresis of DNA Clamps were removed from the cassette. Plate A (Fig. 1) was lifted, starting at the bottom of the cassette containing the high-concentration gel, separating it from the rubber gasket by spatula. Glass plate H, cardboard G and glass plate F were separated from GelBond E, and the gasket was pulled off the GelBond. The bottom triangular section of the gel attached to GelBond was cut off by scissors. The top of the GelBond was trimmed to the width of the ele~trophoresis apparatus (155 mm in application to a BioRad DNA Sub Cell apparatus used). The gel was washed to free it of dye and glycerol either by 3 changes of I I buffer over 48 h, or by 2 changes of 1 1 water followed by 2 changes of buffer over the same period. The washed gel on its GeIBond support was placed into the electrophoresis apparatus and covered with buffer to a height of 2 mm. DNA sample was prepared by diluting 2/~1 of the solution of the 1-kb ladder (1 mg/ml) stored at 4°C with 60 /~1 0.25 x TBE buffer containing 10% glycerol and SPADNS. In some separations (e.g. Fig. 3A), DNA was prelabeled with ethidium homodimer as described [4]. The entire volume was• underlayered into the sample slot using an Eppendorf syringe with Multiflex ~dps. Electrophoresis was conducted at a regulated voltage across the apparatus of 50 V (25 V per 9.8 cm of gel) until the dye had just migrated off the gel. In separations (e.g. Fig. 3B) where DNA had not been prelabeled, the gel with its support was transferred into a tray containing 200 ml of 0.5 /~g/ml ethidium bromide and allowed to stain for 30 min. The wet gel was placed with the GelBond facing upward onto a UV-transilluminator (Fotodyne, New Berlin, WI) the surface of which was covered with SaranWrap to prevent contamination. The
187
gel was photographed using a 2 min exposure and the negative was fixed as previously described [6].
Data processing The photographic negative was viewed by a CCD video camera interfaced with a personal computer as described (see Image acquisition by the computer [1]). Through the interface of that system the Ferguson curves were acquired and digitized. The digitized Ferguson curves were plotted and subjected to least-square linear regression analysis to give values of slope and intercept with the mobility axis at %T = 0 [1].
Results
Transverse agarose pore gradient gel pattern of DNA Transverse agarose pore gradient gel electrophoresis (nominal 0.3-1.3% (Fig. 3A) or 0.2-1.5% (Fig. 3B) SeaKem GTG, 0.5 x TBE buffer, pH 8.4, 20°C) of fluorophore-labeled DNA (1-kb ladder) is shown in Fig. 3. Bromphenol blue (Fig. 3A) or SPADNS (Fig. 3B) was added to the sample and migrated ahead of the DNA zones (the dye was run off just prior to termination of the electrophoretic 0.5% Agarose
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separations shown in the figure). Representative Ferguson curves were correlated with DNA length (bp) as described in Materials and Methods. As a control experiment, a constant 0.5% agarose gel incorporating a 0-20% glycerol gradient was prepared and washed under identical conditions as used for the agarose gradient gels. The 1-kb DNA ladder migrated in this gel as zones parallel to the starting slot (Fig. 4) showing that the glycerol was quantitatively washed out.
Densitornetric gradient profiles of incorporated dye in the hot gradient mixture and in the agarose gel The photographic negative of the cassette containing the hot agarose gradient
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containing a superimposed glycerol-stabilized Bromphenol blue gradient was used for color density acquisition by video camera. The image was digitized and stored in a personal computer. The digitized image was used to compute the agarose concentration in the hot gradient solution (Fig. 5). A conventional densitometry pattern of a representative agarose pore gradient gel of the composition of the one shown in Fig. 3B after overnight gelation through gradual cooling to room temperature is shown in Fig. 6. The densitometric scan was corrected by use of agarose concentration standards incorporating SPADNS to give a scan of increased linearity.
Evaluation of the Ferguson curves formed in agarose gels The Ferguson curves shown in Fig. 3 were acquired in a personal computer as described under Materials and Methods. The digitized curves shown in Fig. 7 were evaluated by least-square linear regression analysis, yielding the slopes, K R, and intercepts on the mobility axis at %T = 0, Yo, depicted in Fig. 8. DNA length (bp) was derived on the basis of a comparison, regarding the relative spacing and intensity of bands, with the gel pattern shown by the supplier. Curves 8A and 8B derived from independent experiments and different concentration ranges of the agarose gradient appear similar, in spite of the difference in labeling.
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Discussion
What is the importance of the transverse agarose pore gradient gel for DNA electropho~esis? The joint report on transverse pore gradient gels formed in polyacrylamide has given a detailed rationale for transverse pore gradient gel electrophoresis in general [1]. In essence, the importance of these gels derives from that of Ferguson plot analysis (reviewed in [10]), which uses electrophoretic mobility values derived
191
from gels of various concentrations to gain insight into molecular conformation and free electrophoretic mobility. As more and more cases of non-linear Ferguson plots become known, and as multi-component DNA systems gain more and more importance, the Ferguson plot analysis required for that purpose becomes excessively laborious and, in case of multicomponent systems, unfeasible without assuming uniformity of net charge density and conformation among all of the components. The transverse pore gradient gel avoids these problems by providing a continuous 'Ferguson curve' over the concentration range of the gradient. In application to DNA, polyacrylamide gels are limited to the DNA length range up to approx. 15 kb; for larger DNA, up to a size of approximately 50 kb, electrophoresis in agarose gels is used [2,11]. Therefore, to potentially secure the benefits of Ferguson plot analysis based on 'Ferguson curves' for the larger size class of DNA, a method of preparing transverse agarose pore gradient gels was developed. Qualitatively, the information with regard to size and conformation (KR) and to free mobility (Yo) from transverse agarose gradient gels corresponds to that described for polyacrylamide in the joint report [1]. However, Ferguson curves are more linear (compare Fig. 3 with Fig. 1 of [1]). Correspondingly, the plot of K R vs. DNA length is convex (Fig. 8) while it is concave when derived from transverse polyacrylamide pore gradient gels (Fig. 3A of [1]). The plots of Yo vs. DNA lengths (Fig. 3B of [1] and Fig. 8) appear indistinguishable, although the paucity of agarose data in the 5 to 10-kb range of DNA precludes a verification in that medium of the concave curvature of the plot. The determination of gel concentration at each point of a transverse pore gradient gel Just as the laboriousness of individual gel formation at many gel concentrations and the uncertainty of relating band positions between those gels is the Achilles' heel of the original Fergu~fon plot method, the pore gradient gel is burdened by the need to ascertain accurately the gel concentration at each point along the pore gradient. Fast-forming polyacrylamide pore gradient gels can be analyzed by densitometry of a dye incorporated into the gradient with the reasonable expectation that dye diffusion is negligible within the short polymerization time, and in gels of relatively high and restrictive gel concentrations [9]. The diffusion of the dye gradient has also been previously neglected in application to agarose gels subjected to densitometry immediately after gelation [3]. However, such an analysis is not optimal since uniformly and fully gelled agarose gels require slow cooling over an extended gelation time, usually carried out overnight. Therefore, an attempt has been made in this study to evaluate the dye gradient prior to gelation, i.e. immediately after gradient formation. This evaluation involved relatively costly instrumentation, viz. a video camera and computer interface for the sake of convenience. More simple photographic and densitometric apparatus could have been used to avoid the error in gel concentration assignment due to overnight dye diffusion. To date, however, this approach to quantitation of %T, using photography of the hot agarose solution prior to gelation of the gradient followed by video acquisition and digitization, has failed as yet to yield an accurate quantitation due
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to our inability with the available setup to eliminate light reflections from the glass surfaces surrounding the cassette; illumination from the back of the cassette also failed. Fig. 5 illustrates the method of documentation of the gradient mixture by photography, video acquisition and digitization, although it was carried out with non-uniform illumination of the cassette. The sigmoidal shape of the gradient resembles that found previously [3]. With the gradient former described, although a linear concentration gradient is theoretically possible, it would in practice only be obtained if the mixing in the syringe was complete before any other solution was displaced in the cassette, i.e. it operated at an infinitely slow flow rate. In reality, complete mixing of the heavy solution into the light solution is never achieved before some of the light solution has been displaced from the syringe, resulting initially in a slightly lower concentration than predicted. As the gradient proceeds, there will be an excess of heavy solution in the mixing syringe, giving rise to a higher concentration than predicted for the latter half of the gradient. Both photography (or video photography) of the gelation mixture and of the gel do not provide a linear response to concentration of the dye and therefore would require internal standards; an array of 7 dye concentration standards in the range of interest can be displayed above the cassette and co-photographed for that purpose, but to date, it remains inaccurate in application to the gradient mixture prior to gelation due to failure to provide a uniform illumination of the cassette (see above). A corresponding correction for the non-linear densitometric response to color intensity is exemplified in Fig. 6. The analysis of the concentration gradient by the optical density of the dye gradient as used in this study gives a continuous profile and appears preferable to gradient analysis by the discontinuous slicing and weighing of the gel (section 2.5 of [12]).
Justification for gel washing While agarose gel gradients over a wide concentration range can be density stabilized without additives [13], pore gradient gels in the range of 0.3 to 1.3% agarose require the addition of a dense medium to the heavy solution, such as the heat-stable Nycodenz [9] or D20 [3]. For making single agarose pore gradient gels the previously used method of density stabilization of the gelation mixture by D20 [3] is preferable to that by glycerol used in the present study since D20 does not increase the viscosity of the gel and therefore does not have to be re/noved prior to electrophoresis. Glycerol was used here, and the additional washing step burdening the procedure was accepted, in view of the long-term goal of preparing batches of multiple pore gradient gels by a single gelation. For a batchwise preparation of these pore gradient gels, it would be possible to monitor on a single representative gel of each batch the gel concentrations within the gradient by dye incorporation into the gelation mixture as in this study, without being forced to conduct the electrophoresis in gels containing the dye, as long as the dye is washed out of the gels prior to electrophoresis. Since for that reason gel washing is desirable, substitution of the costly density stabilizing medium, D20 , by inexpensive glycerol imrJoses no additional burdens on the procedure. The effectiveness of glycerol removal from the agarose gradient gel was demon-
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strated by showing that zones of DNA electrophoresed at a right angle to the orientation of the glycerol gradient were uniformly straight (Fig. 4). More importantly yet, the apparent independence of the migration rate and therefore, effective pore size, on the amount of glycerol present during gelation rules out, at least for the 0.5% gel, a dependence of effective pore size on glycerol within the concentration range used.
Horizontally vs. vertically oriented transverse pore gradient gels The horizontal mode of transverse pore gradient gel electrophoresis has two advantages over the vertical mode described in the joint report [1]: the sample slot replacing the gel surface as the starting zone avoids the necessary unevenness of the latter which is due to the fact that after gelation, spacers need to be removed from it. This unevenness of the starting zone in the vertical orientation perturbs the gel pattern. The second advantage of the horizontal orientation is its compatibility with the widely practiced submarine mode of DNA electrophoresis. Limitations of the procedure In the present study, the required accuracy of the method in defining the agarose concentration has not been achieved and further work is necessary to refine this approach, especially in obtaining uniform illumination conditions for the in situ video photography of the cassette. Furthermore, to become a practical tool for Ferguson plot analysis of DNA, transverse pore gradient gels will have to be produced batchwise. The unit gel of such a batch would be nearly identical to that presented in this study; however, the much increased volume of gel would require a gradient maker with larger volume capacity than the one presently used [7]. Since the precision of gel concentration values within the gradient depends on the precision of gradient formation, a computer-directed gradient maker [14] should be the instrument of choice for batchwise preparation of agarose pore gradient gels.
Simplified description of the method The formation of pore gradient agarose gels oriented at a right angle to the direction of electrophoresis (transverse agarose pore gradient gels) is described. Each component on these gels describes a curve which corresponds to a plot of migration distance vs. gel concentration. The information regarding size and surface net charge of the component and regarding properties of the gel fiber inherent in those curves is evaluated by video acquisition of the gel pattern and its digitization in a personal computer. One important application of the method is in the detection of DNA species and complexes with abnormal conformations and/or free electrophoretic mobilities.
References 1 Wheeler, D., Orban, L., Garner, M.M. and Chrambach, A. (1992) J. Biochem. Biophys. Methods, 24, 171-180.
194 2 SteUwagen, N.C. (1987) In: Chrambach, A., Dunn, M.J. and Radola, B.J., (Eds.), Advances in Electrophoresis Vol. 1, pp. 177-228, VCH, Weinheim, Germany. 3 Gombocz, E. and Chrambach, A. (1991) Electrophoresis 12 (12), (in press). 4 Glazer, A.N., Peck, K. and Mathies, R.A. (1990) Proc. Natl. Acad. Sci. USA 87, 3851-3855. 5 Application Note 324, LKB, Bromma, Sweden. 6 Gombocz, E., Tietz, D., Hurtt, S,S. and Chrambach, A. (1987) Electrophoresis 8, 261-271. 7 Fawcett, J.S., Sullivan, J.V., Chidakel, B.E. and Chrambach, A. (1988) Electrophoresis 9, 216-220. 8 Hook, G.R. and Rasband, W. (1987) In: Bailey, G.W. (Ed.), Proc. 45th Ann. Meet. Electron Microsc. Soc. Am. pp. 920-921, San Francisco Press, San Francisco CA. 9 Tietz, D., Gombocz, E. and Chrambach, A. (1991) Electrophoresis 12, 712-721. 10 Tietz, D. (1988) In: Chrambach, A., Dunn, M.J. and Radola, B.J. (Eds.), Advances in Electrophoresis Vol. 2, pp. 109-170, VCH, Weinheim, Germany.. 11 Orban, L. and Chrambach, A. (1991) Electrophoresis 12, 233-240. 12 Fawcett, J.S., Sullivan, J.V. and Chrambach, A. (1989) Electrophoresis 10, 182-185. 13 Coulson, S.E. and Cook, R.B. (1983) In: Stathakos, D. (Ed.), Electrophoresis '82, pp. 363-370, W. de Gruyter, Berlin, Germany. 14 Altland, K. and Altland, A. (1984) Electrophoresis 5, 143-147.