Enhanced Capacity for Electrophoretic Capture of Plasmid DNA by

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Enhanced Capacity for Electrophoretic Capture of Plasmid DNA by Agarase Treatment of Agarose Gels Kenneth D. Cole* and Bjo¨rn A° kerman Bioprocess Engineering Group, Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, and Department of Physical Chemistry, Chalmers University of Technology, 41296 Go¨teborg, Sweden Received August 4, 2000

Agarase was used investigate the effect of increasing the number of polymer ends on the electrophoretic trapping of circular DNA in agarose gels. The electric field strength required to trap circular DNA was found to be the same in control and treated gels, indicating the treatment did not result in longer traps. Loading experiments indicated that treated gels had a significantly higher capacity for the open circular DNA. Electrophoretic mobility measurements using pulsed fields indicated a higher density of active traps for treated gels compared to controls. Linear dichroism experiments showed that impalement occurred by a fast and a slow process that had characteristic time constants in the one and tens of seconds ranges, respectively. The open circular DNA was more efficiently impaled in the treated gel compared to the control. The considerably higher efficiency of trapping indicated that agarase treatment increased the concentration of traps substantially. Introduction The separation of circular DNA using agarose gel electrophoresis (or by other techniques, such as capillary electrophoresis or chromatography) is essential for monitoring the success of cloning foreign DNA into plasmids, discovering new plasmids, and analyzing the genetic makeup of microorganisms and cellular organelles. An important early study described the size-dependent arrest of the mobility of the open circular (OC) form of plasmid DNA during agarose gel electrophoresis.1 In that publication, the authors did not observe the arrest of the supercoiled (SC) forms of the same plasmids. They proposed that an impalement mechanism was responsible for the arrest of the OC form. Circular DNA can become looped (impaled) over a dangling gel structure (referred to as a trap), and the DNA will be completely arrested if the electric field strength is sufficiently strong to prevent diffusion off the trap. Linear DNA can become looped over the same obstruction, but the molecule will eventually slide off the obstruction when one end wins the tug of war. The use of field inversion gel electrophoresis (FIGE), during which the direction of the electric field is periodically reversed, resulted in the release of arrested OC DNA during agarose gel electrophoresis.2 Periodically turning the electric field off (allowing diffusion in the absence of an electric field) also served to relieve electrophoretic trapping of OC DNA in agarose gels.3 A° kerman4 characterized the electrophoretic traps in polyacrylamide and agarose gels. He observed that both the SC and OC forms of plasmid DNA were trapped at a similar electric field strength in polyacrylamide gels, while only the OC form was trapped in agarose gels at these fields strengths. The structure of an agarose gel is believed to form by the aggregation of agarose double helix polymers into larger

bundles (referred to as suprafibers) resulting in a gel scaffold with relatively open spaces (the pores) between the suprafibers (reviewed in refs 5, 6, and 7). A° kerman4 noted that the differences in the structures of polyacrylamide and agarose gels would explain the differences in trapping of circular plasmid DNA. The diameters of the traps in agarose gels (likely composed of suprafibers) are too large to accommodate the smaller effective diameters of the holes in the SC form, while the polyacrylamide gel traps having smaller diameters can accommodate both SC and OC forms. The structure of agarose gels is influenced by both the conditions used to cast the gel and by modifications of the agarose polymer. Gels cast in low ionic strength buffer or water have smaller pores compared to gels cast in high ionic strength electrophoresis buffers.8-10 Chemical modification of the agarose polymer also influences the pore structure of the gel. Hydroxyethylation of the agarose polymer results in gels that have lower melting temperatures and decreased pore sizes compared to gels cast using unmodified agarose.6,11 Griess et al.12 used electron microscopy to determine the structure of gels made from native and chemically modified agaroses, after reducing the molecular weight (by treatment with γ irradiation). When the molecular weight of chemically modified agaroses was reduced, the resulting gels had increased polymer concentrations in the fibers and larger pores. A basic question that we are posing is if either the physical nature or the number of traps in agarose gels can be changed. The answer to this question has two important implications. The conditions and treatments that change the nature and/or number of traps in agarose gels will give valuable insights into their structure and formation. The second implication is that application of this knowledge would allow investigators to tailor gels for specific separations. In one point of

10.1021/bm005594c CCC: $19.00 © 2000 American Chemical Society Published on Web 00/00/0000

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view, trapping of circular DNA could be viewed as an impediment to the analysis of circular DNA. Trapping limits the size of circular DNA that will migrate in the gel and also the speed at which the analysis can be done (confined to lower electric field strengths). With this view, minimization of the traps would be the desired goal. On the other hand, a gel containing well-characterized traps could be used in a diagnostic manner to rapidly screen for circular DNA above a size limit and for a specific physical form. In this point of view, an investigator could tune a gel for a specific application. In this study we focused on the effect of the agarose polymer molecular weight on the formation of traps. We produced agarose with a reduced average chain length and characterized the traps in the resulting gels. The enzyme agarase was used to reduce the average molecular weight of the agarose polymer. During the course of this study we developed a technique for rapidly measuring the critical electric field strength required to trap circular DNA. This technique allowed the rapid analysis of traps in gels. We used plasmid preparations with three different sizes of DNA, containing both OC and SC forms, to determine the role of size and physical form of the DNA on trapping in these gels. Experimental Section Materials. Agarase I (recombinant), DNA standards, and restriction enzymes were obtained from Life Technologies, Inc. (Rockville, MD).23 DNase I (catalog no. 104 132) was from Boehringer Mannheim (Indianapolis, IN). Agarose (SeaKem LE) was from FMC Corp. (Rockland, ME). Plasmid DNA Samples and DNase I Treatment. The plasmid preparations used in this study were 4.4 kilobase pairs (kb) (pBR 322), 8 kb pairs (pDELTA), and 13.1 kb pairs (pYA101). The contents of SC and OC forms were determined by agarose gel electrophoresis (2 V/cm) for 4 h. The DNA concentrations were determined by fluorescent dye (Hoechst 33258) binding (BioRad, Hercules, CA, US) using supercoiled standards. A bottle of lyophilized DNase I (20 000 units) was gently suspended in 1 mL of 20 mM TRIS, 1 mM MgCl2, pH 7.5. The tube was distributed into small quantities and quick frozen on dry ice and stored at -80 °C. Individual tubes were thawed for use and then discarded. A 5X reaction buffer was prepared consisting of 250 mM TRIS, 50 mM MgCl2, pH 7.5. DNase was diluted by thawing a new tube and adding 10 µL (stock solution) to 10 mL of 1X reaction buffer (1: 1000 dilution). Additional dilutions were also prepared. Typical reactions for each plasmid preparation contained 1.2 µg of the plasmid DNA, 1X reaction buffer, and DNase I dilution in a total of 50 µL. Samples were gently mixed and kept at 21 °C for 1 h. The reaction was stopped by addition of 2 µL of 0.5 M EDTA, pH 8.0. Samples were dialyzed for 4 h against 10 mM TRIS and 1 mM EDTA, pH 7.5. Large Scale Treatment of Agarose with Agarase. Agarose (30 g) was added to 1.2 L of deionized water and brought to a boil with stirring. The solution was stirred for 10 min and then cooled to 43 °C. The solution was adjusted to a pH of between 6.0 and 6.2 using a small amount of

Cole and A° kerman

0.2% acetic acid. Six aliquots (35 mL each) were removed for control samples, and they were boiled for 10 min. Agarase (100 units) was added to the remaining solution (1025 mL), and the solution was shaken (100 rpm) in an incubator at 43 °C for 18 h. The solution was distributed into tubes (35 mL) and boiled for 10 min (to inactivate the enzyme). All of the aliquots were frozen and lyophilized until a constant weight was reached. Apparatus for Transverse Electric Field Gradient Electrophoresis. A commercial submarine gel chamber (CBS Scientific, VWR Scientific, Boston, MA)23 was used. The chamber had an internal cooling chamber, and the buffer solutions were circulated externally through heat exchangers in a water bath. The temperature of the water bath was maintained at 20.0 °C. Gel trays 14 cm wide (12.7 cm inside dimension) by 20 cm long and 2 mm thick combs to form the sample wells were used. An apparatus to generate a transverse electric field gradient was constructed by placing platinum wire electrodes into a gel casting tray. One platinum wire (the negative electrode) was placed parallel to the wells (at a right angle to the sides of the tray). The other electrode (positive) was positioned 3 cm from the negative electrode at one side of the tray and 15 cm from the negative electrode at the other side of the gel tray (forming a diagonal along the gel tray). Gels were cast in an unmodified gel tray and cut into two wedge-shaped pieces. The wedge-shaped gel pieces were positioned in the modified gel tray between the electrodes. Samples were loaded into the wells, and the field was turned on. After the samples had moved into the gel (0.5-1 min), the buffer circulation pump was turned on. A voltmeter with two probes (separated by 1.5 cm) was touched to the gel surface just above the wells to determine the voltage gradient. Electrophoretic Mobility, Pore Size Calculation, and Field Inversion Gel Electrophoresis. Slab gels were run in the submarine mode. The buffer used for mobility measurements was 45 mmol/L TRIS, 45 mmol/L boric acid, and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.3. The electrophoretic velocity was calculated from the distance migrated relative to the starting position (which was determined on an identical but separately stained gel), divided by the running time. To compare the electrophoretic velocities using constant electric fields to those using field inversion gel electrophoresis (FIGE), an effective running time (Teff) was calculated to calculate velocities. This was done by correcting the actual running time (Trun) by the forward pulse times (T+) and the reverse pulse times (T-) using the following expression: Teff ) Trun(T+ - T-)/(T+ + T-)

(1)

Average pore radii were estimated from the velocity of linear DNA in a constant field, using the Ogston model.13 Briefly the pore radius is assumed to be equal to the radiusof-gyration of the DNA which has mobility equal to half the free-solution mobility of double-stranded DNA in the present buffer. Calculations using the Ogston approach are known to produce smaller pore radii results (ca. 4-fold) than more direct methods such as atomic force microscopy (AFM).10 However, this is acceptable for our present

Electrophoretic Capture of Plasmid DNA

purposes where only changes in pore sizes rather than their absolute values are of interest. FIGE was performed using a commercial pulsing instrument (PC 750 Hoefer Scientific Instruments, San Francisco, CA).23 DNA samples were run into the gel (approximately 1 mm) using a direct current field (3.2 V/cm) for 25 min before beginning the pulsing step. Molecular Weight Determination. The molecular weight distributions of agarose (SeaKem LE) and agarase-treated agarose were characterized by gel permeation chromatography and detection using differential refractive index and differential viscometry detection (American Polymer Standards Corp., Mentor, OH) The columns were two AM GPC linear columns with a mobile phase (0.5 mL/min) of dimethyl sulfoxide (DMSO) at 30 °C. Calibration was done using standard pullulans and dextrans dissolved in DMSO and the universal calibration method. The samples and standards were dissolved in DMSO and filtered using a 2.0 micro cup filter before injection. Linear Dichroism. In the linear dichroism (LD) experiments the DNA concentrations were in the range 25-150 µM nucleotides, as determined by absorption at 260 nm and the extinction coefficient 6600 M-1 cm-1. The buffer used for LD measurements was 50 mmol/L TRIS, 45 mmol/L boric acid, and 1 mmol/L EDTA, pH 8.3. Electrophoretic capture of the plasmids was monitored in real time by LD measurement4 in a vertical electrophoresis cell.14 The DNA was introduced into the measuring position in the bulk of the gel by field inversion gel electrophoresis (T+ ) 1 s, T) 0.1 s at 22.5 V/cm), which separated the DNA forms, ensuring that measurements were performed on topologically pure samples. Each DNA type was studied in at least two different and freshly prepared gels. The average temperature in the gel was 20 ( 2 °C as monitored through the electric current. The LD measurements are presented in terms of an orientation factor S, which reflects the average degree of DNA-helix orientation,15 such that S ) 0 for a randomly oriented sample whereas S ) 1 when all DNA molecules are perfectly field-aligned. The growth kinetics of the DNA alignment upon field application was resolved into two additive monoexponential contributions. Linear pYA101 for use in control experiments was obtained by cleavage with XhoI restriction endonuclease. Results Characterization of the Plasmids. Three plasmid preparations were used in this study. All three plasmid preparations contain significant amounts of the OC form in addition to the SC form. The OC form present resulted from nicking of the plasmid during isolation from the host bacteria. The identities of the bands on electrophoresis gels were confirmed by treatment of the preparations with DNase I (Figure 1). Under these conditions, DNase I made single-strand cuts in the DNA. Treatment of the preparations with low concentrations of DNase I resulted in disappearance of the SC form and increased amounts of the OC form. Incubation with increased amounts of DNase I resulted in formation of the linear form and truncated forms seen as a smear below the linear form (Figure 1).

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Figure 1. Identification of the supercoiled and open circular forms of the plasmids used. The samples were run on an agarose gel (1%) at 2 V/cm for 20 min and 4 V/cm for 140 min (20 °C). DNase I treatment was performed as described in the materials and methods section. DNase dilutions were lane 1 (1:500000), lane 2 (1:5000000), lane 3 (1:50000000), and lane 4 (no enzyme). The arrows indicate the OC (open circular), SC (supercoiled), and linear forms of the plasmids.

Agarase Treatment and Characterization of the Agarose. Preliminary experiments were done by adding agarase to molten agarose in a buffer composed of Bis-Tris pH 6.5, near the optimal pH for β-agarase I.16 Gel casts after this treatment had to be soaked in the electrophoresis buffer for extensive periods to equilibrate the gel before electrophoresis. We developed a procedure that avoided the equilibration steps and was suitable for the large-scale treatment of agarose. Agarose was boiled in water, the temperature was reduced to 43 °C, and the pH of the molten agarose was adjusted to approximately 6.2 using a small amount of acetic acid. After enzyme treatment, the enzyme was inactivated by boiling, and aliquots were freeze-dried to a constant weight. The aliquots were stored in the freezer for individual experiments and analysis. Samples of agarose were carried through the same procedure, but not treated with enzyme to provide enzyme blanks. After confirmation that the lyophilization step did not change the properties of the traps, the controls consisted of agarose as obtained commercially. This procedure produced a salt-free powder that could be used to form gels, such as conventional agarose. Samples of treated and untreated agarose were analyzed to determine molecular weights. The samples were dissolved in DMSO to prevent double helix formation.17 The weight average (Mw) and number average (Mn) of the commercial preparation of agarose had values of 119 900 and 50 400, respectively (ratio of 2.4). The agarase-treated preparation had values of 29 200 (Mw) and 5500 (Mn) (ratio of 5.3). The intrinsic viscosities (measured in DMSO at 30 °C) were 245.5 and 56.6 mL/g, for the untreated and treated samples, respectively. The values for the untreated sample were close to the values measured for similar preparations previously measured using aqueous size-exclusion chromatography and low-angle laser light scattering.17 Transverse Electric Field Gradient To Estimate the Critical Electric Field Electric Field Strength Required to Trap Circular DNA. We modified a conventional flatbed electrophoresis apparatus to produce a transverse electric field gradient. This device allowed us to rapidly determine the critical field strength required for immobilization of the circular DNA. The fields that can be applied with this device are quite high, and rapid circulation of buffer was required to remove gases and heat. A thermistor temperature probe,

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Figure 3. Critical field strength required for trapping the OC and SC forms of the plasmids as measured in Figure 2. The measurements for the agarose gels (1%) are the means of at least three determinations and the standard deviations shown by the bars. Agarase-treated gels were composed of 0.5% agarase-treated and 0.5% untreated agarose.

Figure 2. Transverse electric field gradient gel electrophoresis of plasmid DNA. Electrophoresis was in 1% agarose gels at 20 °C as described in materials and methods. (A) Trapping of the OC form of the 13.1 kb plasmid (0.18 µg of DNA applied to each lane). Lane 19 contained the kb ladder plus lambda DNA (48.5 kb). Electrophoresis was for 45 min with a field of 100 V applied at 20 °C. (B) Trapping of the SC forms of the 8 kb plasmid (odd lanes, 0.14 µm) and 13.1 kb plasmid (even lanes, 0.18 µg). Electrophoresis was for 30 min with a field of 100 V applied. (C) Electric gradient measured at 100 and 300 V applied. The fitted curves were second-order quadratics.

inserted into the gel, indicated that the gel temperature only increased approximately 1-2 °C higher than the water bath (20 °C) after electrophoresis for fields up to 300 V. Experiments at 350 V resulted in temperature rises of 2 °C higher at the low field end and 5 °C higher at the high field end. Because of the increasing temperatures, higher fields were not attempted. The transverse gradient gels allowed rapid comparison of many electric field strengths run under similar conditions (shown in Figure 2). The onset of trapping was first seen as a smear and finally as immobilization at the sample wells as the electric field was increased (increasing from the left to right side of gel). Figure 2A shows the onset of trapping of the OC form of the 13.1 kb plasmid and the unhindered migration of the SC form (the fast moving band). Figure 2B shows a gel run at higher electric fields illustrating the trapping of the SC forms of the 8 kb and 13.1 kb plasmids. Note that the OC forms of both plasmids were already trapped at the higher electric fields run for this gel. Figure 2C shows the measured electric field gradient in the gels

shown in panels A and B of Figure 2. Each gel produces a gradient of about 2.5-fold, and combining two gels (cut from the same slab) results in about a 4-fold range of field strength (Figure 2C). The results obtained from the gradient gels are summarized in Figure 3. These results show the molecular weight dependence of the trapping of the OC forms of the plasmids (as previously shown).1 It also shows that the SC forms of the plasmids were trapped, but only at considerably higher electric field strengths compared to the electric fields needed to trap the OC forms. The required electric fields for trapping the SC forms of the plasmids were not as strongly dependent on molecular weight compared to the OC forms. The electric field strength required to trap the SC form of the 4.4 kb plasmid was above 100 V/cm and could not be determined in the current experimental apparatus. Because of the handling involved with the gradient apparatus, it was not possible to do measurements on gels composed entirely of the agarase-treated agarose. This was because of the poor mechanical strength of the gels. The gels fell apart during the handling required for running and staining. A compromise was reached by casting gels composed of 0.5% untreated agarose and 0.5% agarase-treated agarose that could be satisfactorily handled. The results of trapping with these gels were similar to the results obtained with the untreated gels as judged by the onset of trapping and the critical voltage determined for trapping, shown in Figure 3. Loading Experiments. We were able to cast and stain gels composed entirely of agarase-treated agarose when they were cast in a frame of untreated agarose. These gels were stable, if they remained in the casting tray during electrophoresis, staining, and imaging. We used these gels to examine the trapping capacity for the OC form of the 13.1 kb circle. Increasing amounts of the 13.1 kb plasmid were loaded onto gels. The DNA samples were first transported into the gel by use of a low field, and then field strength was increased. The onset of trapping was seen as a smear below the wells (Figure 2). To compare capacity, we picked electric fields strengths that were slightly below the field strength required for trapping for the 13.1 kb OC, trapping occurs at field strengths above 20 V/cm (Figure 3). The loading capacity


Figure 4. Capacity of control agarose (1% on right side) and agarasetreated gels (1%, on left side) for trapping the OC form of the 13.1 kb plasmid at fixed electric field strengths. The DNA loadings were 0.17 µg (lane 1), 0.34 µg (lane 2), 0.68 µg (lane 3), 0.77 µg (lane 4), 1.53 µg (lane 5), and 3.06 µg (lane 6). The kb ladder and lambda DNA were loaded in lanes 7 and 8, respectively. (A) Electrophoresis at 12.8 V/cm for 60 min (20 °C). (B) Electrophoresis at 14 V/cm for 60 min (20 °C). (C) Areas of the smears in the control (1% agarose) gel and agarase-treated gel (1%) run at 14 V/cm shown in B.

gels were run at 12.8 V/cm (Figure 4A) and 14 V/cm (Figure 4B), electric field strengths that are at the onset of trapping (shown by the smears just below the wells). The 13.1 kb SC form was not trapped at these field strengths and migrated unhindered in the gels. The gel made of agarase-treated agarose (left side of panels A and B of Figure 4) had a more compact smear compared to the control gels (right side of the figure) at all of the DNA loading (Figure 4A,B). The


area of the OC smear (from the gel at 14 V/cm) is shown in Figure 4C. The larger area of smearing in the control gels indicated that the control gels had a lower capacity for trapping for the OC form compared to the treated gel. The differences in the areas of the smears between the two gels were not due to reduced diffusion in the treated gel. Specifically the bands of DNA that were not trapped (the SC and linear DNA) are wider in the treated gels, indicating that the treated gels had increased diffusion (dispersion) compared to the control gels (results not shown). The electrophoretic mobility of the linear DNA in the treated gel was also faster compared to the treated gels. Linear DNA traveled approximately 30% faster in the treated gel compared to the control gels. It can therefore be concluded that the narrower zones observed for the smears in the treated gels are due to trapping phenomena and not to differences in diffusion or electrophoretic mobility, which might have resulted from the agarase treatment. Electrophoretic Mobility during Field Inversion Gel Electrophoresis (FIGE). Measurements on the electrophoretic velocity using FIGE were performed on the 13.1 kb plasmid at field strength of 24 V/cm, which was strong enough to capture the OC form of this plasmid but not the SC form (Figure 3). Two types of experiments were performed (Figures 5A,B). In Figure 5A the duration of backward pulse was varied (with a constant forward pulse of 1 s), and in Figure 5B the forward pulse duration was varied (with a constant backward pulse duration of 0.1s). In both cases the velocity of the OC form was affected by the pulse duration, whereas both the linear and SC forms have velocities which were essentially independent of pulse conditions. These mobility measurements were used to probe the mode of trapping in control (1% agarose) and treated gels (composed of 0.5% agarase treated and 0.5% control agarose). In the first case (Figure 5A) the velocity for the OC form is zero for short backward pulses because the field was strong enough to trap the 13.1 kb OC. As the backward pulse duration was increased beyond 3 ms, the velocity increased rather abruptly, because the backward pulses were then long enough to unhook the circular DNA. For even longer reverse pulses the velocity reached a plateau, Vss. Figure 5A shows that the critical backward pulse needed for detrapping was similar in the treated and the control gels (shapes of the curves were the same). By contrast, agarase treatment had a marked effect on the OC velocity behavior in the case of varying forward pulse durations (Figure 5B). In this case the velocity was nonzero for short (forward) pulses because according to Figure 5A the chosen backward pulse duration (0.1 s) was long enough to detrap those molecules that have become impaled during the forward pulse. The pulse-cycle averaged velocity measured in these experiments decreases with increasing duration of the forward pulse for the OC forms because as the forward time is increased the trapped DNA spends a greater fraction of the time trapped. Figure 5B shows that the velocity decreases faster in the treated gel, suggesting that the circles are trapped more rapidly than in the nontreated gel. We also note that for the shortest forward pulse (0.2 s) the velocity was similar in the two gels and close to the plateau velocity

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Figure 6. Field alignment of trapped plasmid DNA. LD response (solid) of 13.1 kb OC form plasmid to a pulse of constant electric field (20 V/cm, dotted) in agarase-treated (0.5% agarase-treated and 0.5% untreated agarose) and control (1% untreated) gel (indicated) at 20 °C. The DNA alignment is given as the orientation factor S(t), where S ) 0 and S ) 1 corresponds to isotropic and perfect DNA alignment, respectively. DNA concentration 75 µmol/L in treated and 77 µmol/L in the control gel.

Figure 5. DNA mobility using FIGE for circular. The pulse-corrected electrophoretic mobility of the indicated forms of the 13.1 kb plasmid was calculated from the distance measured after electrophoresis (20 °C) as described in the materials and methods section. The initial electrophoresis (constant field) used to run the DNA into the gel was at 3 V/cm for 25 min. (A) The electrophoretic mobility versus the reverse pulse times (forward pulse time was fixed at 1 s). (B) The electrophoretic mobility versus the forward pulse times (the reverse pulse time was fixed at 0.1 s). (C) Width of the DNA bands for the FIGE experiments shown in Figure 5A.

in Figure 5A. This suggests that the velocity of DNA in the two gels was similar when under pulsing conditions that allow the molecules to migrate without being trapped. In the experiments with constant forward pulse duration the zone width (Figure 5C) of the OC form passes through

a maximum at approximately the same pulse durations, where the molecules were starting to be detrapped (Figure 5A). By contrast neither the SC nor the linear forms exhibits this maximum. Trapping Kinetics by Linear Dichroism. The velocity experiments were used to examine the effect of the DNA form and size, field strength, and DNA loading on trapping. Such experiments are less suitable for studying the trapping kinetics because the calculated velocity only represents the final result of a chain of events. The trapping process can be followed directly in real time, however, by measurements of DNA alignment.4,18 The electric field readily aligns impaled circles, because the hooking gel fiber acts as a pivot point on which the DNA molecules can swing into the direction of the electric field. Nontrapped circular DNA or linear DNA exhibits much less field alignment because the permanent anchoring offered by the hooking point is missing. The time profile S(t) of the degree of field alignment in the DNA sample can therefore be exploited to monitor the fraction of DNA molecules, which has become trapped as a function of time after the application of an electric field. In particular, the rate of trapping can be followed by analyzing the growth kinetics of the alignment. Figure 6 shows the growth in the DNA alignment in a sample of the OC 13.1 kb plasmid, after a constant field was applied at time zero, with a strength (20 V/cm) high enough to trap the OC form (Figure 3). The slow increase over tens of seconds was specific for the circular DNA because in control experiments the linearized form of the same DNA aligned in less than a second to a level which was about 10 times lower than that for the circle at the same field (results not shown). This is in good agreement with earlier studies of linear and circular DNA in unmodified agarose.4 Figure 6 shows that these characteristics were also present in the treated gel, and that agarase digestion led to a significantly faster growth of the alignment compared to the control gels. In addition, there was a somewhat lower



Figure 7. Semilogarithmic plots of the data in Figure 6. Straight lines are best fits to long-time single exponential.

steady-state value of the DNA alignment than in the control. In both gels, when the field was turned off (at 87 s), the relaxation of the alignment was fast compared to the alignment buildup, another characteristic of impaled circles.4,18 The field-free decay was described by a single exponential and was somewhat faster in the treated gel with a time constant of 0.38 ( 0.01 s compared to 0.52 ( 0.01 s in the control gel. The kinetics of the growth process was focused on because it reflects how efficient a circular DNA was trapped. The semilogarithmic plot in Figure 7 shows that the growth profile was exponential for long times (with a time constant τ2 obtained from the slope of the linear fit) but also that there was a substantial faster component as revealed by the linear fits not passing through the origin. Also the fast component was well described by an exponential (not shown), with a time constant τ1. Thus the overall growth (Figure 6) was biexponential in both gels S(t) ) A1(1 - e-t/τ1) + A2(1 - e-t/τ2)

(2)

where A1 and A2 are the amplitudes of the fast and slow component and A1 + A2 ) Sss, the steady-state orientation factor. The two time constants differed by about 1 order of magnitude (see below), which suggested that there were two distinct types of traps. Interestingly, the agarase treatment affected the long-time linear fits both by increasing the slope and by decreasing the intercept (Figure 7). This means that the growth was faster in the treated gel (Figure 6) for two reasons. The long-term component was clearly faster, but it also played a smaller role, i.e., the fast component made a larger contribution in the treated gel. In this particular case (20 V/cm), the fast component was responsible for 56% of the steady-state value in the nontreated gel but as much as 73% in the treated gel. The faster component dominated the growth at almost all electric fields studied, as demonstrated by the relative amplitude A1/(A1 + A2) of the fast process usually being 0.5 or larger (Figure 8). It was also clear that the fast process increases as the field strength increased. This was true also in the treated gel, to the extent that the fast process made a larger contribution in the treated gel than in the control at all field strengths studied. Figure 9 shows how both time constants decreased with increasing field strength, both in

Figure 8. Relative amplitude A1/(A1 + A2) of the fast alignment component (cf. eq 2) vs electric field strength in agarase-treated (solid) and control (open) gel. A2 is obtained from the intercept of the fitted line in Figure 7 and A1 ) Sss - A2, where Sss is the steady-state orientation factor (cf. Figure 10).

the control and the treated gel. Both time constants were smaller in the treated gel than in the control gel at all fields studied. This suggests that the circles found both types of traps more quickly, i.e., with higher efficiency, in the treated gel. Double-logarithmic plots (insets of Figure 9) show the time constants follow an approximate power law in the field strength, ti ∼ Ea, with exponents a that were significantly more negative than -1 in both gels. Figure 10 shows how the degree of steady-state field alignment (Sss, the plateau in Figure 6) of circular (circle symbols) and linear (triangle symbols) forms depended on the field strength. In both gels Sss for the circles increased sigmoidally, with no significant difference in the behavior between the treated and the control gels. The linear form was considerably weaker aligned because it could not be impaled, as noted above. Linear DNA in this size range was aligned because of the deformation caused by squeezing of the coils during migration through the gel pores.19 Also the alignment of the linear form during migration exhibited a sigmoidal increase,14 but less markedly so. In fact, at low fields, the linear form was more strongly aligned than the OC form, as evidenced by the ratio between the Sss values for linear and OC DNA (see inset of Figure 10) being larger than 1. That the linear form was more strongly aligned at low fields can be understood from the observation14 that for a given field strength the alignment of the linear form increased with DNA length. The OC form had an effective length that was roughly half of that of the linear form of the same molecular weight. It was therefore expected that at fields low enough to allow the OC form to migrate without being trapped, its alignment should be weaker than that for the corresponding linear form. The onset of enhanced circle alignment (S ratio is equal to 1) occurred approximately at 10 V/cm, and the effect saturated at about 15 V/cm. This suggests that the impalement underlying the strong circle alignment began to be important in this field range. Furthermore, it was seen that within experimental uncertainty these field values were the same in the treated and the control gel.

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Figure 9. Time constants τ1 for the fast (A) and τ2 for the slow (B) component of the alignment growth process vs electric field strength in agarase-treated (solid) and control (open) gel. τ2 was obtained from the slope of the linear fit to the long time exponential in Figure 7, and τ1 was obtained from a similar analysis of the semilogarithmic plot of the residual between the raw data (Figure 6) and this long time exponential. Inserts show the double log plots and best-fit lines of these data.

Figure 10. Steady-state alignment obtained from plateau in Figure 6 vs electric field strength, for OC form (circle) in agarase-treated (solid) and control (open) gels, and for linear form in control gel (triangles). Insert shows the ratio of the orientation factors of the linear to the circular DNA values plotted against electric field strength.

Effects of Agarase Treatment on the Gel Structure. The velocity and spectroscopic experiments presented above demonstrated a clear difference in the electrophoretic behavior of circular DNA in the treated and control gels. We therefore investigated differences in structure of the two gels using linear DNA. The mobility of linear DNA was somewhat higher (about 30%) in the treated gel for a given DNA size between 2 kb and 48.5 kb (not shown). With these results the average pore radius (estimated by the Ogston

model as described in the Experimental Section) was 650 Å in the control gel and 750 Å in the treated gel. The migration of long linear DNA such as T2 DNA (164 kb) was a good probe of gel structure, because the wellunderstood migration contains processes on many time and length scales.20 In a constant field the migration occurred by a cyclic conversion between compact and extended states, which was reflected by an oscillatory linear dichroism response to a constant field pulse (Figure 11). It was seen


Figure 11. LD response of linear T2 DNA (166 kb) to pulse of constant electric field of 7.5 V/cm in agarase-treated (0.5% agarasetreated and 0.5% untreated agarose) and control gel (indicated) at 20 °C. DNA concentration 79 µM in treated and 78 µM in control gel.

that T2 DNA migrates in the same oscillatory manner in the treated gel as in the control, so the basic nature of the gel was not affected by agarase treatment. The field-free relaxation was somewhat faster in the treated gel, as it was for the OC form of the plasmid, and the steady-state orientation was lower, both suggesting a somewhat more open gel structure (larger pore size).21 Discussion The field-gradient experiments (Figure 3) show that circular DNA becomes trapped in both types of gels, if the field is strong enough. The alignment kinetics observed in the LD experiments (Figure 6) show that trapping of circles most likely occurs by the same impalement mechanism in both type of gels, as evidenced by a strong and a slow field alignment compared to the linear form of DNA.4,18 On the basis of this premise, the results presented here reveal how agarase treatment affects the properties of the dangling gel fibers, primarily regarding their length distribution and their concentration. When the circular DNA results are interpreted, it should be kept in mind that agarase treatment also makes the gel structure more open. This is clear from the larger average pore size, the higher velocity, and faster diffusion (broader zones) for nontrapped DNA (linear DNA or circular DNA at low fields), and finally the faster field-free LD relaxation of both linear and circular DNA. Agarase digestion does not affect the length of the dangling fibers substantially. The critical field for trapping is not affected, as measured either directly in the field-gradient electrophoresis (Figure 3) or indirectly by the onset of enhanced alignment of circular DNA (Figure 10). The results of the FIGE velocity experiments with constant forward pulse duration can be used to estimate the average fiber length. The backward pulse needed for the net velocity to start to deviate from zero (i.e., to unhook the DNA) can be converted into a distance (i.e., average length of dangling fiber) if the velocity of the circular DNA through the gel is measured. Taking the plateau velocity for long backward pulses as the velocity of the DNA between the traps, the fiber length becomes about 100 nm in both gels.


In contrast, agarase treatment does affect the trapping efficiency at a given field strength, as is suggested by the narrower zones in the loading experiments (Figure 4), the faster decrease in velocity in FIGE experiments with varying forward pulses (Figure 5B), and in the faster trapping kinetics as measured by LD (Figure 6). The narrower zones in the treated gel under trapping conditions (Figure 4) directly demonstrate that the molecules do not have to migrate as far to become trapped in the agarase-treated gel compared to the control gel. This suggests that the agarase treatment increases the density of traps and the trapping capacity. The FIGE experiments (Figure 5B) support this interpretation of the loading experiments (Figure 4) by showing that the DNA molecules have to migrate for longer times in the control gel in order for their (pulse-cycle averaged) velocity to be reduced to the same extent as in the treated gel. Since the velocity between the traps is similar in the two gels (Figure 5A) the longer average time to become trapped in the control gel translates into a longer average distance before a trap is found (i.e., the density of traps is lower in the control gel). The LD experiments (Figure 6) focus on the time needed for trapping rather than migrated distance. These results again reveal a higher density of traps in the treated gel as shown by the faster growth in the LD indicating the circles find the traps (and become field-aligned) more quickly than in the control gel, for a given field strength. The biexponential growth process indicates that both gels contain two types of traps. Before this phenomenon is discussed, a few general remarks about the trapping process should be made. The rate of trapping should increase with increasing field strength in both gels simply because a higher field means a higher search velocity and therefore shorter average time to find a suitable trap. This does not account for all of our results, because this transport mechanism suggests that the LD growth time constants should vary as E-1 (the expected effect of field strength on velocity). The observed dependence is considerably stronger (insets of Figure 9), with power law exponents of -2 to -4. This strong deviation from simple transportdictated kinetics is generally observed for circular DNA alignment in gels4,18 but not for linear DNA, which indeed exhibits exponents close to -1.4,20 This deviation may suggest that the density of effective traps increases with increasing field strength. A higher density of traps at a higher field will occur naturally if there is a distribution of lengths of the dangling fibers: a fiber which is too short to retain a given DNA size (against Brownian motion) at low field will be able to do so at a higher field. Then trapping rates would increase faster with E than search velocity does since the distance to the nearest trap will be shorter at a higher field as new traps become active. The decrease in trapping field with increasing molecular weight (Figure 3) is a reflection of the same effect, because the net force on a larger DNA will keep it impaled on fibers which are too short to trap a smaller DNA. The presence of two contributions to the LD growth suggests there are two types of traps in agarose gels, both with and without agarase treatment. The two types of traps

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may differ in the finding rates, either because there is a higher density of one type of trap, or if the traps are accessible to different extents, perhaps because they are in different parts of the gel. As discussed above, if there is a distribution of trap lengths the effective density of traps increases as the field strength increases. The fact that the faster process (relative amplitude A1) becomes more and more dominant as the field increases (from 40% at 15 V/cm to 70% at 32 V/cm for control gels in Figure 8) therefore suggests that the fast type of traps consists of shorter obstacles on the average, because then they have more to gain from an increase in field. One possibility is thus that the two types of traps correspond to a bimodal distribution in obstacle length, where the shorter types of traps also are found more quickly In the agarase-treated gels both trapping times are shorter, but there is still almost an order of magnitude difference between the time constants. The two types of trapping are presumably similar in nature to those in the unmodified gel since they share several characteristics. The rate of the fast mode is less sensitive to field strength than the slow mode (Figure 9, insets), and the fast mode gains in relative amplitude to a similar extent as in the control gel when the field is increased (Figure 8). It is therefore reasonable to discuss how agarase treatment changes the two types of traps in unmodified gel, rather than assume that the treatment creates a new type of traps. There are two main effects of the agarase treatment on trapping kinetics. The most significant effect is that the fast process consistently has a considerably higher (relative) amplitude by about 40% (Figure 8), so whatever the fast traps are there is a higher fraction of them after agarase treatment. If, as the field dependence indicates, the fast traps are predominantly short, this would support the notion that agarase treatment mainly increases the amount of short traps. Second the agarase treatment speeds up trapping considerably. Both τ1 and τ2 are decreased in the treated gel by about 2- to 3-fold (Figure 9). Both time constants are expected to be shorter because before becoming trapped the DNA migrates faster in the treated gel presumably due to less friction (larger pores). However, the increase in the velocity of linear DNA can only account for a 30% decrease in the time constants and cannot explain a 2-3-fold faster trapping rate compared to the control gel (for both types of traps). A more likely explanation is then that both τ1 and τ2 are shorter in the treated gel because there are more of both types of traps than in the control. We demonstrate here that it is possible to trap supercoiled plasmids in agarose gels. The control and agarase-treated gels both trap the SC forms at the same critical voltages as shown by the gradient gels. The SC forms required significantly higher fields to trap compared to the corresponding OC forms, which indicates that the SC traps are shorter. Trapping studies in polyacrylamide gels4 indicate that supercoiled DNA is more difficult (require higher electric fields) to trap than the OC form because the target holes in the compact SC form are smaller. Thus more slender agarose fibers are needed to trap the SC form. Such fibers will also be shorter (or more flexible). However, we cannot rule out

that the same traps could accommodate both OC and SC forms. Cone-shaped traps that only allow the SC forms to access the outer portions of the trap would give the same results. As mentioned in the Introduction the gelling conditions and chemical modifications of agarose can determine the final structure of the resulting gel. Gels cast in low ionic strength buffers or in water have smaller average pore size.7-10 We cast gels in water and soaked them in electrophoresis buffer for prolonged periods. Such gels had smaller pore sizes (shown by reduced electrophoretic mobility of linear DNA), but these gels showed no significant difference in the critical voltage to trap OC plasmid DNA as measured in the transverse gradient gels (results not shown). Chemical derivation of the agarose polymer also has a significant effect on the structure of the resulting gels.8 Hydroxyethylated agarose is commercially available as a low melting point agarose (SeaPlaque, FMC Bioproducts, Rockland, ME).23 These gels also did not have significantly longer traps when screened using transverse electric field gradient gels (results not shown). Hydroxyethylated agarose can be used to selectively purify circular DNA, but the separation is not dependent upon the electric field strength, and this suggests another separation mechanism besides the field-dependent impalement mechanism.22 We have been successful in creating longer traps as demonstrated by decreasing the critical voltage required for trapping by adding additional polymers to agarose gels (in preparation). We are in the process of characterizing the resulting traps. This study shows that we were successful in increasing the number of traps in agarose gels by using agarose polymers with more ends. The physical properties of these gels were significantly different that the control gels. The treated gels were poor in terms of their electrophoretic performance with linear DNA and mechanical strength. The treated gels had larger pores but gave poorer resolution of high molecular weight linear DNA. These gels, poor performers for linear DNA, illustrate that the molecular weight of the agarose polymer is a powerful variable for changing the density of the traps for circular DNA in agarose electrophoresis gels. References and Notes (1) Mickel, S.; Arena, V.; Bauer, W. Nucleic Acids Res. 1977, 4, 14651482. (2) Levene, S. D.; Zimm, B. H. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4054-4057. (3) Serwer, P.; Hayes, S. J. Electrophoresis 1987, 8, 244-246. (4) A° kerman, B. Biophys. J. 1998, 74, 3140-3151. (5) Serwer, P. Electrophoresis 1983, 4, 375. (6) Kirkpatrick, F. H. OVerView of Agarose Gel Properties; Kirkpatrick, F. H., Ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1990; Vol. 9, pp 9-22. (7) Serwer, P.; Griess, G. A. J. Chromatogr. B 1999, 722, 179-190. (8) Griess, G. A.; Guiseley, K. B.; Miller, M. M.; Harris, R. A.; Serwer, P. J. Struct. Biol. 1998, 123, 134-142. (9) Waki, S.; Harvey, J. D.; Bellamy, A. R. Biopolymers 1982, 21, 19091926. (10) Malloum, M.; Pernodet, N.; Tinland, B. Electrophoresis 1998, 19, 1606-1610. (11) Greiss, G. A.; Moreno, E. T.; Easom, R. A.; Serwer, P. Biopolymers 1989, 28, 1475-1484. (12) Griess, G. A.; Edwards, D. M.; Dumais, M.; Harris, R. A.; Renn, D. W.; Serwer, P. J. Struct. Biol. 1993, 111, 39.

Electrophoretic Capture of Plasmid DNA (13) A° kerman, B. Phys. ReV. E 1996, 54, 6685-6696. (14) Jonsson, M.; Akerman, B.; Norden, B. Biopolymers 1988, 27, 381414. (15) Norde´n, B.; Elvingson, C.; Jonsson, M.; A° kerman, B. Q. ReV. Biophys. 1991, 24, 103-164. (16) Morrice, L. M.; McLean, M. W.; Williamson, F. B.; Long, W. F. Eur. J. Biochem. 1983, 135, 553-558. (17) Rochas, C. Carbohydr. Res. 1989, 10, 289-298. (18) A° kerman, B. J. Phys. Chem. B 1998, 102, 8909-8922. (19) Magnusdottir, S.; A° kerman, B.; Jonsson, M. J. Phys. Chem. 1994, 98, 2624-2633.

Biomacromolecules, Vol. 1, No. 4, 2000 781 (20) A° kerman, B. Electrophoresis 1996, 17, 1027-1036. (21) A° kerman, B.; Jonsson, M.; Norden, B.; LaLande, M. Biopolymers 1989, 28, 1541-1571. (22) Upcroft, P. Gene 1988, 65, 319-323. (23) Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedures. Such identification does not imply recommendation by NIST, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

BM005594C

Enhanced Capacity for Electrophoretic Capture of Plasmid DNA by

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