Miniaturized System for Rapid Field Inversion Gel Electrophoresis of

Feb 11, 2010 - Separation conditions: 1.0 wt % certified low range ultra agarose, Eavg = 2.19 V/cm, Ef = 8.75 V/cm, Er = 4.375 V/cm, τf = τr = 400 m...
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Anal. Chem. 2010, 82, 1831–1837

Miniaturized System for Rapid Field Inversion Gel Electrophoresis of DNA with Real-Time Whole-Gel Detection Airong Li,†,‡ Xiaojia Chen,‡ and Victor M. Ugaz*,‡ College of Chemical Engineering, Sichuan University, Chengdu, PR China, and Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas, 77843-3122 Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (∼10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60-90 min. The compact size of the device also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields. The ability to perform size-based separations of DNA fragments greater than ∼5 kilobases (kb) in length is important in many analytical and preparative applications.1-3 Gel electrophoresis is typically used to perform these separations, but the same operating conditions that are optimal for analysis of shorter DNA (i.e., * To whom correspondence should be addressed. Phone: (979)458-1002. Fax: (979)845-6446. E-mail: [email protected]. † Sichuan University. ‡ Texas A&M University. 10.1021/ac902490e  2010 American Chemical Society Published on Web 02/11/2010

constant electric fields) are generally not able to provide sufficient resolution as fragment lengths extend into the kilobase range.4,5 This situation arises as a consequence of an unfavorable transition to an extended molecular conformation during migration through the sieving matrix, resulting in a relatively weak scaling of mobility with DNA fragment size.6 A variety of approaches have been explored to overcome this limitation by altering the characteristics of DNA migration through the gel so that size-dependent molecular motion can be recovered.7-24 Pulsed field gel electrophoresis (PFGE) methods, whereby the electric field direction is periodically changed, have proven to be particularly effective and are among the most widely used methods for separation of large DNA fragments. In contrast to DNA migration in continuous fields, the periodically changing electric field direction introduces a reorientation process characterized by its own size dependent time scale (i.e., larger DNA fragments take longer to realign their migration with the new field direction and therefore travel a shorter distance during each pulse interval).24,25 In this way, PFGE (1) Slater, G. W.; Desruisseaux, C.; Hubert, S. J.; Mercier, J.-F.; Labrie, J.; Boileau, J.; Tessier, F.; Pepin, M. P. Electrophoresis 2000, 21, 3873–3887. (2) Slater, G. W.; Guillouzic, S.; Gauthier, M. G.; Mercier, J.-F.; Kenward, M.; L.C., M.; Tessier, F. Electrophoresis 2002, 23, 3791–3816. (3) Viovy, J. L. Rev. Mod. Phys. 2000, 72, 813–872. (4) Boyd, B. M.; Prausnitz, J. M.; Blanch, H. W. Electrophoresis 1998, 19, 3137– 3148. (5) Holmes, D. L.; Stellwagen, N. C. Electrophoresis 1990, 11, 5–15. (6) Madden, T. L.; Deutsch, J. M. J. Chem. Phys. 1991, 94, 1584–1591. (7) Birren, B. W.; Hood, L.; Lai, E. Electrophoresis 1989, 10, 302–309. (8) Birren, B. W.; Lai, E. Nucleic Acids Res. 1994, 22, 5366–5370. (9) Chen, N.; Wu, L.; Palm, A.; Srichaiyo, T.; Hjerten, S. Electrophoresis 1996, 17, 1443–1450. (10) Chu, B.; Wang, Z. J. Non-Cryst. Solids 1991, 131, 685–692. (11) Gekeler, V.; Weger, S.; Eichele, E.; Probst, H. Anal. Biochem. 1989, 181, 227–233. (12) Griess, G. A.; Rogers, E.; Serwer, P. Electrophoresis 2001, 22, 981–989. (13) Griess, G. A.; Serwer, P. Appl. Phys. A: Mater. Sci. Process. 2002, 75, 223– 228. (14) Heller, C.; Pakleza, C.; Viovy, J. L. Electrophoresis 1996, 16, 1423–1428. (15) Heller, C.; Pohl, F. M. Nucleic Acids Res. 1989, 17, 5989–6003. (16) Kirkpatrick, F. H.; Dumais, M. M.; White, H. W.; Guiseley, K. B. Electrophoresis 1993, 14, 349–354. (17) Shikata, T.; Kotaka, T. Macromolecules 1991, 24, 4868–4873. (18) Stellwagen, J.; Stellwagen, N. C. Electrophoresis 1989, 10, 332–344. (19) Vollrath, D.; Davis, R. W. Nucleic Acids Res. 1987, 15, 7865–7875. (20) Wagner, L.; Lai, E. Electrophoresis 1994, 15, 1078. (21) Wang, M.; Lai, E. Electrophoresis 1995, 16, 1–7. (22) Doggett, N. A.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1992, 20, 859–864. (23) Sobral, B. W. S.; Atherly, A. G. Nucleic Acids Res. 1989, 17, 7359–7369. (24) Woznicki, A.; Greger, J. Biochemistry 1993, 32, 7181–7185. (25) Mathew, M. K.; Smith, C. L.; Cantor, C. R. Biochemistry 1988, 27, 9210– 9216.

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methods have greatly extended the range of DNA fragment lengths that can be separated from 10s of kilobases up to 10s of megabases in size. Although these advancements have enabled widespread adoption and commercialization of PFGE technology, the method still suffers from inherent drawbacks including extremely time-consuming separations (running times of 10 h or more are typical) and a slab-based format where analysis is performed by photographing a poststained gel upon completion of the run.7-9,15,17,19,20,26,27 These issues impose severe limitations on the ability to characterize fundamental physical parameters associated with DNA migration under pulsed fields (e.g., to establish the size dependence of fragment mobility and diffusive broadening of the separated zones). This kind of information is critically needed to rationally identify which combinations out of the total ensemble of variable parameters (electric field strength, duration, direction, etc.) are most likely to yield optimal separation conditions. Many previous efforts to extract such data have focused on field inversion gel electrophoresis (FIGE), a subset of PFGE methods whereby the electric field direction alternates between an angle of 180° (i.e., the forward and reverse directions of DNA migration). But the cumbersome and time-consuming nature of the experiments make it impractical to explore a wide range of parameters. Consequently, the body of available data is restricted to fragment mobility (extractable from a single experiment) with no characterization of diffusion (requiring multiple experiments to establish the rate of band broadening).7,23,27 Both of these parameters play a key role in determining the achievable separation resolution. We have previously explored the potential for miniaturized slab gel instruments to address the need for improved characterization of DNA migration phenomena during PFGE.28 One of the major advantages associated with moving to a compact device format is that much faster separation times are achievable (i.e., 1-2 h), thereby making it practical to perform a larger ensemble of experiments. We demonstrated this in the case of FIGE by exploring the effects of electric field pulsing parameters on separation resolution and by investigating the size dependence of DNA fragment mobility for fragment lengths ranging from 2.5 to 35 kb. These data helped to provide a more detailed picture of the factors influencing separation performance, but the instrument design still imposed limitations. For example, while the use of a fluorescence microscope-based imaging system allowed migration of prestained DNA to be observed during the separation, it also proved cumbersome because it did not allow the entire gel to be imaged within the field of view. This introduced variability between successive scans of the gel that made it difficult to reliably track broadening of the migrating bands, ultimately preventing us from obtaining diffusion measurements. A further limitation of our previous design was the use of a switching power supply to modulate the electric field. Although this simplified the apparatus, it did not allow variation in applied voltage waveforms and introduced a short delay during switching between the forward and reverse field directions. Here we describe a greatly improved miniaturized apparatus for performing FIGE based DNA separations. Although the basic design builds on our previous work, the new system we have (26) Krawczyk, M. J.; Pasciak, P.; Dyadejczyk, A.; Kulakowski, K. Acta Phys. Pol., B 2005, 36, 1653. (27) Sabanayagarn, C. R.; Holzwarth, G. Electrophoresis 1996, 17, 1052–1059.

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assembled incorporates significant modifications that greatly expand its capabilities. First, we have simplified the gel imaging process in a way that eliminates the need for a fluorescence microscope. Instead, a compact UV lamp is used to illuminate the electrophoresis device from below so that it can be imaged from above with a CCD camera. The gel box incorporates a quartz base to maximize UV light transmission so that the entire gel can be imaged at a very high resolution. Second, we have replaced the switching power supply previously used with a combination of a function generator and voltage amplifier that allows more precise control over the voltage pulse sequence waveform. This combination of features enables the separation process to be monitored during the entire run, with complete separations of a 2.5 kb dsDNA ladder achievable in 60-90 min (an order of magnitude faster than conventional instruments). More importantly, the ability to obtain sequential high-resolution images of the separation process makes it possible to, for the first time, quantitatively characterize diffusive broadening of the migrating zones. We harness these capabilities to systematically study the effects of experimental parameters so that a deeper understanding of DNA migration in pulsed fields can be obtained. The results of these experiments also provide a rational basis to identify optimal separation conditions. EXPERIMENTAL SECTION The experimental setup consisted of three primary components: (1) a miniaturized slab gel-based FIGE device, (2) a power supply composed of a function generator and high voltage amplifier to run the separation, and (3) a gel imaging system incorporating an integrated UV illumination source and CCD camera. The entire setup is straightforward to assemble and operate, creating a simple yet powerful platform to enable rapid electrophoretic separations while allowing the migrating DNA zones to be continuously monitored throughout the experiment. Miniaturized FIGE Device. Electrophoretic separations were performed using a miniaturized slab gel device consisting of Plexiglas strips mounted on the surface of an optically transparent base using 30 min epoxy (Devcon, Danvers, MA) (Figure 1). Strips 8 mm tall were positioned to define an outer buffer reservoir, and 5 mm tall strips created an inner gel casting area and support for mounting platinum electrodes. Platinum wire (28 gauge; ScienceKit, Tonawanda, NY) was threaded through holes drilled at both ends of opposing inner Plexiglas strips so that the electrodes could extend along the entire gel width (4 cm spacing between electrodes). This configuration is based on a design we described previously,28 with the exception that a quartz base (3 × 3 × 1/16 in. quartz plate, cat. no. CGQ-0620-10; Chemglass, Vineland, NJ) was substituted for the glass base used in the original design. We found that this critical modification was needed because UV absorbance from the original glass base plate did not provide sufficient transmitted intensity to excite the fluorescently labeled DNA. Pulse Wave Generator. A second key modification to the electrophoresis apparatus consisted of replacing the switching power supply used previously with a function generator capable of producing arbitrarily shaped voltage waveforms at frequencies up to 20 MHz (Model 33220A; Agilent Technologies, Inc., Santa Clara, CA). The function generator output served as input to a high voltage amplifier providing a 50x gain (Model 603; Trek, Inc.,

Figure 1. Miniaturized FIGE system consisting of a miniaturized slab gel device, power supply driven by a function generator and high voltage amplifier, and gel imaging system with illumination source and CCD camera. This system permits the entire gel to be photographed at high resolution during the course of the separation run so that the migration of fluorescently labeled DNA can be recorded and analyzed to extract mobility and diffusion for each band in a single experiment.

Medina, NY). The amplifier output was then connected to the electrodes in the electrophoresis device. An oscilloscope was used to monitor a reference signal from the high voltage amplifier that was proportional to the output. Fluorescence Imaging. The final important component of the experimental setup was the gel imaging system. Although the fluorescence microscope used in our previous design allowed the motion of the migrating DNA zones to be recorded, only a small region of the gel could be observed within the field of view. We overcame this limitation by modifying a conventional UV gel illumination system (ChromaDoc-It; UVP, Upland, CA) so that the entire gel area in our FIGE device could be imaged. This system consisted of an enclosure and mounting stage for a 12.1 megapixel digital camera (PowerShot G9; Canon, Inc., Tokyo, Japan). The ChromaDoc-It system provides built-in gel illumination from above, however we found that this configuration produced undesirable reflections from the gel surface and did not excite sufficient fluorescence from the migrating DNA. Greatly improved results were obtained by illuminating the gel from below using a compact UV lamp (Model UVL-21 (4 W, 365 nm); UVP, Upland,

CA) placed on the floor of the ChromaDoc-It enclosure so that the light was directed upward toward the camera. The slab gel device was then placed directly on top of the lamp (the 4 W lamp intensity was found to deliver the best trade-off between sufficient illumination to excite the fluorescently labeled DNA while minimizing background fluorescence from the gel). The supplied emission filter mounted in front of the digital camera was replaced with a 520 nm band-pass filter (cat. no. NT43-173; Edmund Optics, Inc., Barrington, NJ). The camera was connected to a computer so that image capture could be automated and controlled using the supplied DigiDoc-It LS software. The following set of camera settings were consistently applied in all experiments: shutter speed ) 13 s; aperture ) 2.8; CVB ) fluorescent; quality ) superfine; resolution ) large; AF distance ) zone focus (close-up); lighting mode ) bright (default). All images were acquired using the maximum optical zoom level. Gel Casting. Gels were prepared by mixing appropriate amounts of agarose powder (Certified Low Range Ultra Agarose unless otherwise noted, Bio-Rad, Hercules, CA) with 1x TBE buffer (Extended Range TBE, Bio-Rad, Hercules, CA). Molten agarose was dispensed into the inner gel casting area of the FIGE device using a 1000 µL pipettor (a total volume of 3 mL was dispensed), and a homemade comb cut from a thin plastic sheet was used to define the sample loading wells (each well was approximately 1 mm wide, 2 mm long, and 2 mm deep). The gel was cured at room temperature for approximately 30 min, after which the comb was removed and the ends of the gel were gently separated from contact with the electrodes using a thin blade (this permitted dissipation of bubbles generated at the electrodes). The resulting slab gel was approximately 3 mm thick. DNA Sample Preparation. FIGE separations were studied using a double-stranded DNA standard ladder sample (2.5 kb Molecular Ruler (cat. no. 170-8205, Bio-Rad, Hercules, CA)) containing 14 bands ranging from 2.5 to 35 kb in length spaced by 2.5 kb increments. Fluorescently prestained DNA samples were prepared by combining 7 µL of the DNA ladder with 3 µL of a 100x solution of SYBR Green I (Invitrogen/Molecular Probes, Carlsbad, CA) and 1 µL of 6x Orange Loading Dye (Fermentas, Hanover, MD). Approximately 1 µL of the DNA sample was loaded into each well (3-4 wells were run in each experiment). Electrophoresis Procedure. After the DNA samples were loaded, a 0.5× TBE running buffer solution was added into the outer buffer reservoir until the liquid level just covered the top surface of the gel. The platinum electrodes were then connected to the power supply and the FIGE device was placed inside the gel illumination enclosure. We found that the favorable combination of a compact gel size, low voltage, and short running times minimized temperature changes during the run. Consequently, all separations were performed at room temperature without active temperature control. The gel was photographed at multiple times during the course of the run (typically at 15 min intervals) for subsequent analysis. Data Analysis. Analysis of the gel images was performed using previously described methods.28-31 Mobility measurements (28) (29) (30) (31)

Chen, X.; Ugaz, V. M. Electrophoresis 2008, 29, 1–7. Lo, R. C.; Ugaz, V. M. Electrophoresis 2006, 27, 373–386. Lo, R. C.; Ugaz, V. M. Lab Chip 2008, 12, 2135–2145. Ugaz, V. M.; Burke, D. T.; Burns, M. A. Electrophoresis 2002, 23, 2777– 2787.

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were extracted from the acquired gel images using the freely available software package ImageJ (http://rsbweb.nih.gov/ij/) to determine distances traveled by each DNA band. The migration velocity of each band was then computed from the resulting distance versus time data. Under a constant electric field E, electrophoretic mobility µ is defined as µ ) L/Eτ, where L is the distance traveled by the migrating DNA zone during time τ.32 In pulsed field gel electrophoresis, however, the applied voltage is periodically varied so that mobility must be expressed in terms of an average applied electric field (µ ) L/Eavgτ). Considering the case of FIGE where the electric field direction periodically alternates between the forward and reverse directions of DNA migration, we define an average electric field strength as Eavg ) (Efτf - Er τr)/(τf + τr). Here, τf and τr represent the respective times over which forward and reverse electric fields of strength Ef and Er are applied. Mobility data obtained from three parallel gel lanes were averaged. Diffusion coefficients were calculated to quantify the rate at which the migrating DNA zones experienced broadening during the separation run.29,33-35 Assuming a Gaussian zone shape, a diffusion coefficient D can be related to the standard deviation σ of the mass distribution within the DNA band after an elapsed time τ according to D ) σ2/2τ. Values of σ were extracted using image analysis code written in MATLAB (The Mathworks, Novi, MI) that applied a Gaussian fit to the fluorescence peaks within a user-defined region of interest. Plots of σ2 versus run time were then constructed so that D could be determined from the slope of a linear fit to the data. Diffusion coefficient values obtained from three parallel gel lanes were averaged. In general, diffusion measurements are challenging to obtain because they strongly depend on the ability to resolve small changes in the shape of the DNA zones as they experience spreading (this sensitivity is reflected in the larger error bars, as compared with the mobility data). These measurements are also very time-consuming to perform using conventional benchtop slab gel instruments because of the need to perform multiple separation runs. Consequently, we are not aware of any published data characterizing band broadening in PFGE under a wide range of conditions. The high resolution images obtainable in our experiments combined with fast separation times (∼90 min) allow us to overcome these limitations so that the process can be characterized in an accurate and reproducible way. The separation resolution R is a parameter that quantifies the ability to distinguish neighboring electrophoretically migrating zones. It is defined as the ratio of the distance between adjacent bands to sum of their half widths at the base. If the migrating DNA zones are assumed to follow a Gaussian profile, then the half width at the base of each peak can be taken as twice its standard deviation, yielding an expression of the form R ) (x2 x1)/2(σ1 + σ2), where (x2 - x1) is the distance between the two peaks. This calculation is straightforward to perform since the (32) Crater, G. D.; Gregg, M. R.; Holzwarth, G. Electrophoresis 1989, 10, 310– 315. (33) Krawczyk, M. J.; Dulak, J.; Pasciak, P.; Kulakowski, K. Electrophoresis 2004, 25, 785–789. (34) Pasciak, P.; Krawczyk, M. J.; Gudowska-Nowak, E.; Kulakowski, K. J. Biol. Phys. 2005, 31, 365–373. (35) Pasciak, P.; Kulakowski, K.; Gudowska-Nowak, E. Acta Phys. Pol., B 2005, 1737–1743.

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Figure 2. Evolution of a typical separation run in the miniaturized FIGE system shows the ability to resolve DNA fragments ranging from 2.5 to 35 kb in 90 min. The separation conditions used were determined to be optimal based on our experiments (see the Supporting Information): 1.0 wt % certified low range ultra agarose, rectangular waveform, Eavg ) 2.19 V/cm, Ef ) 8.75 V/cm, Er ) 4.375 V/cm, τf ) τr ) 400 ms, run time ) 90 min. Images of the gel are acquired at 15 min intervals and subsequently analyzed to determine mobility and diffusion of each migrating band. The loading wells at the top of each lane are 2 mm wide.

band widths are directly obtainable from Gaussian fits to the intensity data applied during our diffusion analysis. Here, we plot resolution data with respect to the peak associated with the shorter fragment (e.g., a data point at 10 kb indicates resolution between the 10 and 12.5 kb peaks). RESULTS AND DISCUSSION Separation Performance of the Miniaturized FIGE System. The systematic studies enabled by the miniaturized FIGE system make it possible to clearly identify the effects of key operating parameters on mobility, diffusion, and separation resolution. These data permit rational selection of conditions (agarose gel and fluorescent dye formulations, electric field shape and pulse parameters) that provide an optimal balance between separation speed (high mobility), sharpness of the migrating bands (low diffusion), and range of fragment lengths that can be separated (a detailed description of these studies is provided in the Supporting Information). Based on these results, we identified optimal operating conditions for separation of double-stranded DNA fragment sizes up to 35 kb, as shown in Figure 2 depicting the progression of a typical electrophoresis run. After injection into the gel, DNA migration proceeds rapidly such that nearly all bands in the ladder (up to 35 kb) can be clearly distinguished in 90 min. This contrasts sharply with the 15 h running time recommended by the ladder’s manufacturer to achieve comparable results. In addition to rapid separation, the ability to acquire snapshots of DNA migration in the gel at any time during this period makes it possible to quantitatively extract mobility and diffusion coefficient measurements for each band in the ladder. The order of magnitude reduction in separation time is a result of a favorable interplay among several unique factors associated with the miniaturized slab gel format. The compact device size provides the benefit of inherently improved heat transfer and lower voltages to achieve electric field strengths comparable to those used in conventional benchtop instruments. This greatly minimizes

Figure 3. DNA separation results under optimal conditions in the miniaturized FIGE system (corresponding gel images are shown in Figure 2). Mobility versus fragment size data are plotted on both (a) linear and (b) log scales to highlight regimes characterized by different size scalings (slopes are indicated on the plot in (b)). Data are also plotted for (c) diffusion coefficient and (d) separation resolution with three characteristic regimes of behavior indicated. Separation conditions: 1.0 wt % certified low range ultra agarose, Eavg ) 2.19 V/cm, Ef ) 8.75 V/cm, Er ) 4.375 V/cm, τf ) τr ) 400 ms, run time ) 90 min.

Joule heating effects and promotes a highly uniform temperature profile within the matrix. Additionally, the small size of the sample loading wells enables the DNA to be injected into the gel as a narrow and highly focused zone so that adjacent bands can be resolved in a shorter separation distance. Finally, the function generator driven power supply provides more precise control of the electric field than a conventional high-voltage switching apparatus. We have found separation reproducibility and robustness to be excellent in the system’s intended analysis range (e.g., fragment lengths up to the size of λ-DNA). This is an important size regime in many applications (analysis of restriction digest products, cloning vectors, etc.), and we have routinely used the device in this way in our own lab. Analysis of DNA Migration During FIGE. Several key trends emerge when corresponding size dependences exhibited by the DNA mobility, diffusion, and separation resolution are plotted in Figure 3. First, the mobility data are characterized by an expected decrease with increasing DNA fragment length (Figure 3a). However there is also evidence of changes in slope occurring within the 10-20 kb fragment size range. This behavior becomes more clearly apparent in a log-log plot of mobility versus DNA size (Figure 3b), where zones with the following scalings can be distinguished: µ ∼ M-0.20 (smallest DNA size; 2.5-10 kb), µ ∼

M-0.16 (transition region; 10-15 kb), and µ ∼ M-1.22 (largest DNA size; 27.5-35 kb). The limited data available for FIGE separations in range of DNA fragment length generally show scaling exponents in the vicinity of -0.4 to -0.5 (see Table 1 in ref 28); however, they are obtained over a much broader range of DNA size and are not subdivided according to the zones identified here. The diffusion data follow a surprising and much different trend characterized by three distinct regimes of behavior with respect to DNA fragment length (Figure 3c): an increase in diffusivity for fragments shorter than ∼10 kb (regime I), followed by a decrease to a local minimum between 10-20 kb (regime II), followed by an increase for fragments ∼20 kb and longer (regime III). The interplay between the size dependent scalings of mobility and diffusion has interesting consequences on separation resolution, which first decreases with increasing DNA length (also evident by a zone of slight band compression near 10 kb, see the gel images in Figure 2), then increases over an intermediate range fragment size range, and ultimately decreases again for the longest fragments (Figure 3d). Moreover, the boundaries between these three regimes approximately correspond the DNA size ranges where different size dependences are observed in the diffusion data. The intermediate range of increasing separation resolution Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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with DNA length (of interest from a practical standpoint) appears to arise primarily as a consequence of the local decrease in diffusivity, a phenomenon that to our knowledge has not been previously reported. We propose a possible interpretation of these observations in terms of a transition from the shortest DNA fragment lengths (