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J. Phys. Chem. 1996, 100, 3252-3263
DAPI Staining of DNA: Effect of Change in Charge, Flexibility, and Contour Length on Orientational Dynamics and Mobility of the DNA during Agarose Gel Electrophoresis Anette Larsson, Bjo1 rn A° kerman, and Mats Jonsson* Department of Physical Chemistry, Chalmers UniVersity of Technology, S-41296 Gothenburg, Sweden ReceiVed: June 7, 1995; In Final Form: September 19, 1995X
Microscopy studies of the DNA electrophoresis process demands fluorescent staining of DNA, and it is important to reveal how the staining affects the electrophoretic behavior. In this work the influence of the dye DAPI on the behavior of DNA in agarose gel has been studied by mobility and orientational measurements. From the measurements and comparisons with theories, it is estimated that DAPI, at the binding ratio 0.2 dye molecule per DNA base, decreases the electrokinetic charge and persistence length of DNA by 13% and at least 40%, respectively, and increaes the contour length by 20%. These changes affect strongly both mobility and orientational dynamics of the DNA, but the mode of motion is not affected for neither short nor long DNA (2-164 kpb). For long DNA that reptates with oscillations between stretched and coiled states, the steady-state mobility in 1% agarose is reduced by 30% at all studied fields (5-25 V/cm). In these fields the characteristic times in the buildup of the steady state orientation as well as the reorientation time and the period time in the oscillatory motion are increased by 60%. For both DAPI-DNA and uncomplexed DNA the field-free relaxation of the major part of the orientation is dominated by two fast processes with time constants that are similar for the two molecules but with a ratio between the amplitudes that is different. Analysis of this difference indicates that the lower mobility of the DAPI-DNA complex is due mainly to the strong reduction in the persistence length caused by the dye. However, the difference in the orientational dynamics during migration and in field-free relaxation disappears if comparison is made, not at the same field strength, but at field strengths where the two molecules show the same mobility.
1. Introduction The research on the mechanisms behind the separation of DNA in pulsed field gel electrophoresis has been intense since the introduction of the technique.1,2 The details of how DNA moves and interacts with the gel are still, however, far from fully understood although there is known today from both experimental and theoretical studies that stretching, orientation, and reorientation of DNA are key factors in the separation. (For recent reviews see refs 3-5.) Spectroscopy utilizing polarized light and fluorescence microscopy are important and complementary tools in the experimental studies. Fluorescence microscopy demands, however, optical probing of the DNA, and it is therefore important to reveal how the probing affects the electrophoretic behavior of the molecule, also in view of the fact that electrophoresis normally is made on native DNA. From the results of the spectroscopic6-13 and fluorescence video microscopy14-19 studies and computer simulations,20-24 it has been shown that the DNA in a constant field not simply migrates in an oriented state, as obtained by the early reptation models,25-27 but instead oscillates between stretched and compact conformations, a mode of migration which is also observed in extended reptation models.28 The study of the orientation and the orientational dynamics in both constant and pulsed fields and how they correlate with the migration velocity is therefore of great importance for revealing the mechanisms behind the separation. Orientational spectroscopy has the advantage, compared to the video microscopy technique, that it can follow and quantify even very fast processes (down to the millisecond range). Linear dichroism (LD) and birefringence measurements have shown that the relaxation of the orientation when the field is turned off contains fast components, suggesting * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, January 15, 1996.
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that internal modes of the chain within the tube have to be taken into account.8,9,11-13 DNA is flexible on the length scale of the gel pores (200 nm29), which is outside the resolution of the microscope (about 250 nm30), and local stretching or compression on this scale is probably essential for the separation. Furthermore, the spectroscopic techniques give orientational data that represent the average behavior of all molecules in the sample, and the measured quantities can therefore be of direct value for selection of experimental conditions that give optimal separation. However, video microscopy has demonstrated a complicated inter- and intramolecular distribution of the orientation, and it is clear that a detailed understanding of the electrophoresis process also requires knowledge about this distribution. Orientation spectroscopy, for obtaining a quantitative description of the average behavior, and fluorescence microscopy, for obtaining a qualitative picture of single events, are therefore a powerful combination in the study of the way DNA moves through the gel. Fluorescence microscopy demands that the DNA is probed with a fluorescent dye. The fluorescent probes used in most studies of DNA behavior in gel electrophoresis have been ethidium bromide (EB),14,18 4,6-diamidino-2-phenylindole (DAPI),15 and acridine orange (AO).16 According to reviews over the use of fluorescence microscopy in DNA studies, DAPI gives the highest fluorescence intensity of these and also, in the presence of mercaptoethanol, low fluorescence fading and low risk for strand breaking.30,31 Recently, the dimer YOYO of the dye oxazole yellow has also been used as a DNA probe in video microscopy studies and been found to give excellent contrast in the pictures due to its very strong fluorescence when bound to the DNA,32-34 but so far no systematic study has been made of the rate of fading and DNA breaking for this dye. Since electrophoretic separations are made on pure DNA and since bound dyes may lead to changes in the electrophoretic © 1996 American Chemical Society
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charge, length, and flexibility of the DNA and to a changed interaction with the gel, it is important to determine to what extent the hydrodynamic and conformational behaviors of the probed molecules differ from that of native DNA. Reese has investigated the effect of the intercalator ethidium bromide by comparing the mobility of native and probed DNA.35 The LD technique developed in our laboratory for measurement of electrophoretic orientation in gels exploits the dichroic absorption of the DNA bases;7 i.e., it can be used with or without optical probes and thus also in an investigation to what degree the probing alters the average orientational dynamics of the DNA. In this paper we present results from a study of how the mainly groove bound DAPI affects the orientational dynamics and mobility of T2 DNA, which has a size that force the molecule to migrate by reptation through the gel, and also how the dye affects the mobility of smaller DNAs (fragments of λ-DNA Hind III digest) run under such conditions that the migration is more “Ogston-like”. 2. Materials and Methods 2.1. Linear Dichroism. Linear dichroism (LD) is defined as the difference in the absorption of light polarized parallel and perpendicular to a given laboratory axis through the sample:
LD(λ) ) A|(λ) - A⊥(λ)
(1)
where λ denotes the wavelength of light. On a molecular level the absorption process is determined by a vectorial property known as the transition dipole moment, the absorption being maximum when the electric vector of light is polarized parallel to the transition moment and zero when perpendicular to it. LD can therefore provide information about molecular orientation when the transition moments are known. In the absence of orientation in the sample A| ) A⊥ and LD ) 0. If the molecules are oriented, the orientation can be characterized by the reduced linear dichroism (LDr) defined as36
LDr(λ) ) LD(λ)/Aiso(λ)
(2)
where Aiso denotes the absorbance of the corresponding isotropic sample. The LDr factorizes into a product between an optical factor, O, and an orientation factor, S.36
LDr(λ) ) SO
(3)
O is related to the effective angle between the transitions moments contributing at the wavelength λ and the principal orientation axis of the molecule (in our case the DNA helix axis), whereas S describes the average orientation of this axis relative to the reference direction. S is 0 in a sample where the molecules are randomly oriented and 1 in a sample where they are perfectly oriented parallel to the reference direction. With the conditions prevailing in our experiments the doublestranded DNA has been in its B-form conformation. For pure DNA S can then be determined directly from eq 3 by measuring LDr of the base absorption band at 260 nm and by using an optical factor of -1.48.36 However, with DAPI bound to DNA S cannot be determined since the directions of the DAPI transitions (absorption around 360 and 260 nm) in the complex are uncertain and since its transitions in the UV region overlap the DNA transitions. It has thus not been possible to quantify the effect of DAPI on the degrees of orientation but only on the orientational dynamics, which in this work is directly given by the time behavior of the LD since there has been no change in Aiso during the relatively short electrophoretic pulses applied in the experiments.
2.2. Buffer, DNA, and DAPI. A TBE buffer containing 50 mM Tris base, 50 mM boric acid, and 1.25 mM EDTA (pH 8.3) was used throughout. The T2 DNA (164 kbp), which was used for the gel LD experiments, was kindly provided by Ms. Chantal Turmel, Centre National de Reserche, Montreal, Canada. It was enclosed in 0.5% agarose plugs. The T2 used in all other experiments was from Sigma. (According to the manufacturer, more than 80% of the preparation migrate as a single band in field inversion gel electrophoresis.) For the mobility experiments the DNA was used to form sample plugs of 1% agarose. The λ-DNA Hind III digest was from Pharmacia. The digest contains a series of fragments in sizes ranging from 0.1 to 23.1 kbp. However, since the fragments are present in equimolar amounts, only the longer bands in the gel (23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kbp) contained enough material to be accurately measured. The digest was heated to 65 °C for 5 min and quickly cooled on ice prior to gel loading to break the annealed sticky end that forms between the 23 and 4 kbp fragments upon prolonged storage. DAPI (4′,6-diamidino-2-phenylindole) was from Serva and has been used without further purification. It is a dicationic dye, which binds reversibly to double-stranded DNA. At the DAPI-DNA base ratio used in this work (see Results and Discussion) the binding is heterogeneous with binding modes that depend on the base sequence. In AT regions the dye is believed to bind in the minor groove of the DNA37 whereas in GC regions it is belived to be intercalated.38 2.3. Gel Preparation. Gels, 0.15-1% (w/w) agarose (DNA ultrapure from Pharmacia), were cast in the TBE buffer. The gel solutions, prepared by weighing buffer and agarose powder, were heated to boiling for 5 min and subsequently corrected for evaporation by addition of water. The solutions were allowed to cool to 50 °C and then poured into the casting frames of conventional horizontal submarine electrophoresis cells. The gels were allowed to solidify for at least 1 h, whereupon they were submerged in the TBE buffer or, in experiments where the effect of DAPI on DNA was to be studied, in the TBE buffer containing 10 µM DAPI. In the latter case the gel was equilibrated with the TBE-DAPI solution before the electrophoresis was started. The DAPI concentration in the solution after the equilibration has in all experiments been close to 8 µM and has also been constant during the electrophoresis, as determined from light absorbance at 345 nm by using the molar absorptivity 27 000 M-1 cm-1.39 A corresponding determination of the DAPI concentration in the gel has not been possible due to the turbidity of the gel, but it is reasonably to assume that it has also been constant and equal to that in the overlayered solution. The possibility that DAPI is adsorbed to the gel was checked by submerging 20 × 10 × 10 mm3 gel pieces (made with TBE buffer) in 2 mL of TBE buffer containing various concentrations of DAPI. After 24 h the DAPI concentrations in the surrounding solutions were determined by absorption at 340 nm and were compared with the DAPI concentration of a control solution without a gel (to account for DAPI adsorption to the tube walls). After correction for a small swelling of the gel pieces (5% in linear dimension), the partition coefficient for DAPI between gel and buffer was found to be 0.52, 0.53, and 0.54 for final DAPI concentrations of 8, 16, and 32 µM, respectively; i.e., a slight preference for the gel is observed. 2.4. Mobility Measurements. Mobility measurements in constant fields were performed in a Biorad Sub-Cell DNA electrophoresis cell. Ten gels were prepared in parallel lanes by using Plexiglas spacers glued to the gel tray; each gel was approximately 15 × 1.5 × 0.3 cm3. Identical samples were
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loaded into the wells of the eight central gels; the gels closest to the walls of the cell were not used because the temperature of these was found to differ from that of the central ones during the electrophoresis. In runs where all gels in the cell were identical (0.8% or 1% agarose, respectively) the electrophoresis was stopped at regular intervals, and at each stop two of the gels were removed for analysis, and replaced by premoulded gels equilibrated against the same buffer as used in the experiment, which was found necessary to keep the distribution of the electric current and thus the temperature constant during the whole electrophoresis. In runs on pure DNA the removed gels were soaked for 1 h in buffer solution containing ethidium bromide and then rinsed for 1 h with buffer before they were transferred to a UV transilluminator and photographed with a Polaroid camera. In runs where DAPI was present no staining with ethidium bromide was necessary; on transillumination the bands were easily seen due to the strong fluorescence from the DNA-DAPI complexes. Low concentrated gels are difficult to handle and keep in place in the gel tray. Electrophoresis in gels of concentration lower than 0.8% was therefore performed by embedding the gels in the cell in frames of 0.8% agarose moulded at the edges of the tray; one of these frames also contained the sample wells. Gels of four concentrations were run in each experiment, and since in this case the gels could not be removed, the analysis was instead made by transferring the gel tray with the gels to the transilluminator after completed electrophoresis. In all runs the temperature has been kept at 22 ( 2 °C by circulating the electrophoresis buffer through an outer heat exchanger in a thermostating bath. The circulation also secured a constant electrolyte concentration along the cell. The field strength in the gels was determined by a probe consisting of two electrodes 4 cm apart; fluctuation in the field between the gels was less than 2%. Distances were measured from the sample wells to the center of the migrating bands, and they were found to increase linearly with time at all gel concentrations. The given mobilities are averages from two to four experiments. 2.5. Electrophoretic LD Measurements. LD experiments have been performed on T2 DNA in 1% agarose gels. The spectrophotometer and the electrophoresis cell used for the measurements have been described in detail earlier.40 The spectrophotometer is constructed to measure LD by phase modulation technique (50 kHz) and has a vertical light beam so that measurements on a horizontal gel can be made. The gels used in these experiments were 10 × 10 cm2 and 2 mm thick. To secure that the DNA at the measuring position in the gel was homogeneous, it was migrated to this position by pulsedfield (120°, 20 s pulse time, 5 V/cm) electrophoresis in a Pharmacia-LKB Pulsaphore electrophoresis cell before the gel was transferred to the electrophoresis chamber of the spectrophotometer. All LD measurements have been performed as described in ref 40 and at a temperature equal to that in the mobility experiments (22 ( 2 °C). Square-formed electrophoretic field pulses were fed to the cell from the power supply via a twochannel pulse aggregate (Pharmacia-LKB) with rise and decay times less than 50 µs. The LD signals were recorded on a Nicolet 2090 oscilloscope. Presented data are averages of four to six measurements. The averaged LD relaxations were normalized between zero and unity and analyzed by fitting to a double-exponential decay
LD(t) ) Ae-t/τ1 + Be-t/τ2 + C
(4)
The quality of fitting was estimated from the residual between the fitted and the measured curve. To obtain good fits, the
constant term C had to be added, but it never accounted for more than 12% of the total LD amplitude. The fitting was performed by means of the computer program ASYST 2.1 using a standard routine within the program for exponential curve fitting. 2.6. Determination of the DAPI-DNA Binding Ratio. The amount of DNA present in the electrophoretic bands in the gels has varied from experiment to experiment. The DAPIDNA binding ratio (the number of DAPI molecules bound per DNA base) should still be same in all experiments since the association constant of DAPI is high, and the free DAPI concentration has been held constant and equal in all experiments. DAPI is an achiral molecule but exhibits circular dichroism (CD) when bound to DNA, and it has been shown that the binding ratio can be determined from the CD spectrum of the complex.41 It has not been possible, however, to measure accurately the CD of the complex when in the gel due to the background CD from the gel itself. We have instead imitated the gel experiments, where DAPI was introduced in the gel by equilibration with the overlayered TBE-DAPI solution, in equilibrium dialysis experiments where dialysis tubes containing DNA solutions of varying concentrations were equilibrated with the TBE-DAPI solution. The experiments were performed so that the equilibrium DAPI concentration outside the tubes was the same (8 µM) as that in the overlayered solution in the gel experiment. Since DAPI adsorption to the gel is very weak, this situation should well mimic the gel experiments. The DNA was T2 DNA, and the DNA concentrations were 0.02, 0.10, and 0.15 mM phosphate, respectively, covering the interval within which the DNA concentration has varied in the gel experiments. The equilibration was allowed to take place during 24 h at 20 °C, and CD spectra of the DAPI-DNA solutions were then measured on a Jasco J-500 spectropolarimeter using a 1 cm quartz cell. 2.7. Flow LD Spectra. To get a notion of how DAPI is bound to the DNA under our experimental conditions, flow LD spectra of the DAPI-T2 DNA solutions from the dialyses (see above) were measured and compared with the flow LD spectra of pure T2 DNA solutions (of the same DNA concentration) and with earlier published spectra on DAPI-DNA. The molecules were oriented in shear flow fields generated in a Coutte cell.36 LD was measured on a Jasco J500 spectropolarimeter equipped with a quarter wave device as described elsewhere,36 and the isotropic absorbance was measured on a Cary 2300 spectrophotometer. For the DAPI-DNA complexes the reduced LD spectra were calculated as LDr ) LD/Aiso, after correcting the isotropic absorbance for the contribution from free DAPI. 3. Results 3.1. DAPI-DNA Binding Ratio. Figure 1 shows CD spectra of the DAPI-DNA solutions from the equilibrium dialysis experiments which was performed to estimate the DAPI-DNA binding ratio in our experiments (see Materials and Methods). The spectra are normalized with respect to the DNA concentration. They are recorded in the 300-450 nm region of DAPI where Norde´n et al.41 have shown that the CD of DAPI-DNA varies with the binding ratio. At low binding ratios their CD shows a maximum at 335 nm with a shoulder at 365 nm, but with increasing amount of associated DAPI the CD is redistributed to give, at a binding ratio of 0.2, a CD maximum at 375 nm, a weak shoulder at 340 nm, and a new negative, very weak band in the long-wavelength wing of the absorption band (at 410-430 nm). The negative band was interpreted as due to DAPI-DAPI interactions (exciton coupling), indicating a DAPI binding close to saturation. The shape
DAPI Staining of DNA
Figure 1. Circular dichroism (CD) spectra of DAPI-T2 DNA complexes from equilibrium dialysis experiments (see text). DNA concentrations in bases are (a) 15, (b) 10, and (c) 2 mM. Equilibrium free DAPI concentration (outside the dialysis bags) is 8 µM. Optical path length in the CD measurements is 1 cm.
Figure 2. Flow reduced linear dichroism (LDr) spectra of (a) DAPIT2 DNA complex (solution a, Figure 1) and (b) pure T2 DNA of the same concentration. Optical path length ) 0.1 cm. Shear gradient ) 470 s-1.
of our CD spectra is almost identical with this spectrum except at the lowest DNA concentration (Figure 1, spectrum c) which differs at the wings of the absorption band (no “exciton” band can be seen for instance). However, at this concentration the noise was larger than the signal in these wavelength regions, and the resemblance as to the rest between the spectra suggests that the binding of DAPI to DNA is independent of the DNA concentration under the experimental conditions used in our study. The CD maximum in our spectra is at 371 nm, indicating a binding ratio somewhat less than 0.2. The binding of DAPI to DNA is dependent on the base sequence of the DNA since both the association constants and the binding modes differ for AT and GC sequences.38 Our dialysis experiment was performed on T2 DNA, but in the gel experiments we have also used fragments from a Hind III digest of λ-DNA. Norde´n et al. used calf thymus DNA in their study, but in view of the great resemblance of the CD features, and the fact that natural DNA contains all four bases in similar proportions, it is still reasonable to assume that the binding ratio in our gel experiments have been the same, and close to 0.2, for all DNAs. 3.2. Flow LD Spectra. In Figure 2 the reduced linear dichroism spectra obtained by flow orientation of one of the dialyzed DNA-DAPI samples (0.15 mM DNA-phosphate) is compared with the LDr spectrum of native DNA oriented by the same flow gradient. The LDr of the native DNA is negative and flat around 260 nm (where the DNA bases absorb), as expected for B-form DNA.36 The LDr amplitude is a measure
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Figure 3. Typical LD signals from (a) DAPI-T2 DNA (LD 360 nm) and (b) T2 DNA (LD 260 nm) in 1% agarose to an electrophoretic field pulse the direction of which is changed 90° when the molecules have reached their steady-state orientation. The signals are normalized with respect to this steady-state value. The step-formed curve in the bottom part of the figure shows turn-on (at 0 s), change of direction (at 12.5 s), and turnoff (at 25 s) of the electric field (field strength 10 V/cm). The times to reach the overshoot, tp, the undershoot, tu, and steady state, tss, in the buildup of the orientation and the time to reorient after the field direction is changed, treo, are defined at curve b. LD reference axis: initial field direction. For DAPI-T2 DNA the normalized LD at 260 nm (not shown) is identical, but with opposite sign, to that at 360 nm.
of the degree of helix orientation, which depends on DNA length and stiffness.36 For the DNA-DAPI complex the LD in this wavelength region is also negative, but with a superimposed contribution from bound DAPI, due to a transition (centered at approximately 260 nm) which has less negative LDr than the DNA bases.42 The fact that the LDr amplitude is decreased over the whole 260 nm band indicates, however, that the binding of DAPI reduces the degree of DNA orientation. The positive LDr spectrum in the DAPI absorption region (320-450 nm) shows that a majority of the DAPI molecules are groove bound42 since binding along the groove makes the transition moments responsible for absorption around 360 nm to lie more parallel than perpendicular to the helix axis. 3.3. Electrophoretic LD Measurements. Figure 3 shows LD signals from T2 DNA and T2 DAPI-DNA, respectively, in 1% agarose when the molecules start to migrate and orient in a constant electric field (field strength 10 V/cm), the direction of which is then changed 90° when the steady-state LD has been reached. The signals are normalized with respect to this steady-state value to better illustrate the difference in the orientational dynamics of the two systems. LD is in both cases given with the initial electric field direction as reference axis. For the uncomplexed DNA we have used the LD of the DNA bases at 260 nm and for the DAPI-DNA complex the LD of DAPI at 360 nm (only the bound DAPI contributes to the LD) to determine the average orientation of the DNA helix relative to this direction. The orientational responses start off from the DNA systems in their relaxed states. Both systems display an over- and undershoot in the buildup of the steady-state orientation. The LD is negative for native DNA which shows that the helix axis is oriented in the direction of the field (see Materials and Methods). From the positive electrophoretic LD in the DAPI absorption band, the same conclusion can be drawn for DAPI-stained DNA, since flow LD shows that the LD of the DNA and DAPI bands have opposite sign under our binding conditions. After the change in field direction, the LD in both cases decreases in magnitude, passes zero, and finally reaches a steady level of opposite sign (positive for DNA and negative for DAPI-DNA) but of the same magnitude as in the first field
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Figure 4. (a) Plots of tp, tss and (b) of tu, and treo vs field strength for pure T2 DNA (open symbols) and T2 DAPI-DNA (filled symbols). The symbols are tp (0), tu (4), tss (O), and treo (3). The insert in (b) is a double-logarithmic plot of tp for pure T2 DNA (0) and T2 DAPIDNA (9). The lines are least-squares fits. Agarose concentration ) 1%.
pulse which shows that the molecule has now reached the same degree of orientation, but with the helix axis along the new field direction. The influence of the field strength on the kinetics of the buildup of the steady-state orientation as well as of the reorientation is illustrated in Figure 4 where the times to reach the overshoot, undershoot, and the steady state (tp, tu, and tss, respectively) and the reorientation time (treo) are plotted as functions of the field strength for both DNA and DAPI-DNA. The figure shows that all the times decrease with increasing field strength and that they are throughout longer for DAPIDNA than for the uncomplexed DNA, i.e., the binding of DAPI to DNA reduces the rate both in buildup of the orientation and in reorientation. As seen in Figure 3, for both DNA and DAPI-DNA the LD relaxation when the field is turned off consists of a fast process accounting for more than half of the relaxation, followed by a slower process. Fitting to a double-exponential decay (eq 4) was found to give a satisfactory description of the relaxations if an offset term was included, as judged from the residuals between the fitted and the measured curves (not shown). Figure 5a shows that the two LD relaxation times, τ1 and τ2, are independent of the field strength and also have the same values for both DNA and DAPI-DNA, although a certain reservation must be made for the longest time where the data are rather scattered. The relative amplitudes A and B, corresponding to the time constants τ1 and τ2, are shown in Figure 5b for both DNA and DAPI-DNA. 3.4. Mobility Measurements. 3.4.1. Dependence of the Constant Field Mobility on the DNA Size. Figure 6 shows how the constant field mobility of DNA, with and without bound DAPI, varies with the size of DNA in 1% agarose gel and at a field strength of 10 V/cm. The DNAs were chosen to cover the DNA size region where the separation of pure DNA is lost in constant field electrophoresis, i.e., the region where the mobility changes from being size dependent for the smaller
Larsson et al.
Figure 5. Electric field strength dependence in (a) the relaxation times τ1 (O) and τ2 (0) and (b) the corresponding relative amplitudes A and B of the field-free relaxation of the steady-state orientation (from fits according to eq 4). Filled symbols represent DAPI-T2 DNA and open symbols T2 DNA. Agarose concentration ) 1%.
Figure 6. Constant-field mobility of DNA (0) and DAPI-DNA (9) of various DNA sizes: T2 DNA (164 kbp), T7 DNA (37.8 kbp), Hind III fragments of λ-DNA (23.1, 9.4, 6.6, 4.3, 2.3, and 2.0 kbp). Agarose concentration ) 1%. Electric field strength ) 10 V/cm.
DNAs to becoming essentially independent of size for the larger ones. The boundary between the two regimes is determined by the gel concentration and the field strength, and the change has been interpreted as a transition from sieving motion to reptation motion. It can be seen from the figure that the binding of DAPI to DNA does not affect the position of the transition boundary at the gel concentration and field strength used in our experiment. The binding leads to a mobility decrease, however, of about 30% independent of the DNA size. The weak adsorption of positively charged DAPI to the gel (section 2.3) will give rise to an electroosmotic flow in the same direction as the DNA migration (toward the anode), so that the measured electrophoretic mobility of the DNA-DAPI complexes is higher than if measured in a native gel. The direct effect of the binding of DAPI on the DNA mobility is thus an even stronger decrease than the 30% that results from our comparison with nonstained DNA in a native gel. The effect can be expected to be weak, however. In native gels electroos-
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Figure 8. Electric field strength dependence in constant field mobility of T2 DNA (0) and T2 DAPI DNA (9). Agarose concentration ) 1%.
range 1-25 V/cm during constant field electrophoresis in 1% agarose gel. It is seen that the mobility for both pure and complexed DNA increases with the field and that the mobility for the complex throughout is lower than for pure DNA. Figure 7. Ferguson plots of (a) DNA and (b) DAPI-DNA of the DNA sizes 9.4 (0), 6.6 (O), 4.3 (4), and 2.3 kbp (3). Electric field strength ) 1.27 V/cm. The lines are least-squares fits. Mobility is in cm2/(V s).
mosis toward the cathode is induced by anionic sulfate and pyruvate groups,43 which are present in typically millimolar concentrations44 and which in extreme cases may affect the DNA mobility by 20-30%.45 The effect on the DNA mobility from electroosmosis due to adsorbed DAPI in micromolar concentrations can therefore be expected to be almost negligible. 3.4.2. Constant Field Mobility as a Function of Gel Concentration. Ferguson Plots. The Ogston sieving theory describes the electrophoretic behavior of globular undeformable molecules of a size smaller or comparable to the gel mean pore size.46 A plot versus the gel concentration of the logarithm of the electrophoretic mobility (Ferguson plot)47 of such a molecule is linear with a slope (retardation coefficient, K) that is proportional to the particle size and an ordinate intercept at zero gel concentration that is equal to the logarithm of the free mobility of the particle.48,49 There is no theory of such plots for a fibrous molecule like DNA, but at low electric fields and low agarose gel concentrations small DNAs give linear Ferguson plots, which has been taken as an indication that the DNA molecules then migrate as globular coils that are sieved by the gel according to the Ogston mechanism.50 To investigate whether DAPI affects the coil size and free mobility of DNA, the mobility of the Hind III fragments in the absence and presence, respectively, of DAPI were determined at some low gel concentrations (0.15-0.8%) and at a low electric field (1.27 V/cm). In the Ferguson plots straight lines (least-squares linear regression) were found to fit the data only for gel concentrations
