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Nov 24, 2013 - However, conventional gel electrophoresis loses its efficiency for DNA molecules larger than ∼20 kilo base-pairs (kbp).(11) pulsed fi...
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High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis? Lei Liu,† Vijaykumar Veerappan,‡,⊥ Qiaosheng Pu,§ Chang Cheng,∥ Xiayan Wang,*,† Liping Lu,† Randy D. Allen,‡ and Guangsheng Guo*,† †

Department of Chemistry and Chemical Engineering, Beijing University of Technology, Beijing 100124, China Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, United States § Department of Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China ∥ Analytical Department, Albany Molecular Research, Inc., Rensselaer, New York 12144, United States ‡

S Supporting Information *

ABSTRACT: A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 105 plates for the longest (48.5 kbp) and 3.7 × 105 plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsedfield gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

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sample volumes.14 These limitations led researchers to seek alternative methods for efficient separation of high molecular weight DNA samples. Recent advancements in micro/nano electromechanical systems have led to the fabrication of wellorganized artificial structures to mimic sieving matrices in microchips to resolve DNA molecules. These artificial structures include entropic traps,15,16 nanoslits,17−19 nanochannels,20−22 micro/nano pillars,23,24 nanopores,25−27 and other structures.28−32 Although, these techniques provide faster analyses and reduced sample volumes, limited resolution continues to constrain the practical applications of these devices. Most recently, our group has made efforts to improve chromatographic DNA separations. We have developed a novel

odern technologies for separating DNA fragments with high speed and high resolution is critical for the advancement of molecular biological and genomics research. High-performance liquid chromatography (HPLC) and electrophoresis are the two primary techniques for DNA separations. Different HPLC methodologies, such as ion-pair reversed-phase LC,1−3 size-exclusion chromatography,4,5 slalom chromatography,6−8 and hydrodynamic chromatography9,10 have been employed, but the low resolving power of these methods prevents HPLC from competing with gel electrophoresis for DNA separation. As a result, agarose gel electrophoresis is most frequently used. However, conventional gel electrophoresis loses its efficiency for DNA molecules larger than ∼20 kilo base-pairs (kbp).11 pulsed field gel electrophoresis (PFGE), which was developed in the early 1980s, is almost exclusively used for resolving large DNA molecules (≥10 kbp).12,13 While it can achieve high resolutions for large DNA separations, PFGE is a tedious and time-consuming assay that requires large © 2013 American Chemical Society

Received: October 4, 2013 Accepted: November 24, 2013 Published: November 24, 2013 729

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DNA with YOYO-1 (a fluorescent intercalating dye) at a dyeto-base pair ratio of ∼1:10 in 1× TE buffer. The final total DNA concentrations were 60 ng/μL for the Lambda DNA Mono Cut mix and 70 ng/μL for Lambda Mix Marker 19. The dsDNA samples were freshly prepared right before use. Arabidopsis BAC DNA Preparation, Restriction Digestion, and Agarose Gel Electrophoresis Separation. An Arabidopsis plant BAC clone T6B20 carrying 104.9 kbp of genomic DNA insert from Arabidopsis (Columbia ecotype) chromosome II was obtained from Arabidopsis Biological Resources Center (Columbus, OH). BAC plasmid DNA was prepared using Qiagen plasmid midi kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. BAC Plasmid DNA concentration was estimated using NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). A 5 μg sample of BAC DNA was incubated with 20 units of SacII restriction endonuclease (New England Biolabs, Beverly, MA) overnight at 37 °C. To inactivate the restriction enzyme, the sample was incubated at 65 °C for 20 min and then stored at −20 °C until further use. A 200 ng of SacII digested T6B20 BAC plasmid DNA was separated on 0.7% agarose gel containing 0.5 μg/mL ethidium bromide using 1× TAE buffer (40 mM Tris-acetate and 1 mM EDTA at pH 8.3) at 3 V/cm. Lambda Mix Marker 19 (Fermentas Inc., Glen Burnie, MD) was used for comparison. Kodak gel documentation system was used to analyze the DNA fragments. Rice BAC DNA Preparation and Restriction Digestion. A Rice BAC clone OSJNBa0088H09 containing a 145.391 kbp plasmid was a gift from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences (Beijing, China). BAC plasmid DNA was prepared using the Large Amount Plasmid Preparation kits (Aidlab, Beijing, China), according to manufacturer’s instructions. BAC plasmid DNA concentration was measured by BioPhotometer Plus (Eppendorf, Germany). The BAC plasmid DNA was digested with restriction enzyme PmeI (NewEngland Biolabs, Beverly, MA) at 37 °C for 3 h and then the enzyme was heat inactivated by incubation at 65 °C for 20 min. Pulsed Field Gel Electrophoresis Separation. The PmeI digested Rice BAC plasmid DNA was separated by PFGE in a 1.0% agarose gel in 0.5 × TBE buffer (1X = 89 mM Tris base, 89 mM boric acid, 2 mM Na2EDTA) with a CHEF Mapper XA (Bio-Rad) at 6 V/cm and 14 °C for 15 h with a ramped pulse time from 0.5 to 1.5 s. Lambda DNA Mono Cut mix (New England Biolabs, Beverly, MA) was used for comparison. Upon completion of electrophoresis, the agarose gel was stained with ethidium bromide and destained in distilled water. Gel was visualized under UV light, and a digital image was obtained using a Kodak gel documentation system. Measurement of Eluent Flow Rate. An empty 20 μm i.d. capillary was connected via a union to the outlet of a 5 μm i.d. capillary, and the moving meniscus of the eluent in the 20 μm i.d. capillary was monitored under a microscope. The flow rate was calculated according to

hydrodynamic chromatographic (HDC) approach using narrow bare open capillary for both DNA and protein separation at high efficiencies.33−37 Major advantages of this approach include simple and inexpensive apparatus, low sample and reagent consumption, high efficiency and resolution, long column lifetime, and elimination of sieving matrices. In this work, we demonstrated particularly the high efficiency and resolving power of this approach for separating kilo basepair DNA fragments. The HDC chromatograms revealed percentage resolutions of less than 9.0% in general for the Lambda DNA Mono Cut Mix. We also evaluated HDC performances and its basic HDC parameters for these analytes. We further utilized this method to resolve restriction enzyme digested real-world samples [Arabidopsis and Rice bacterial artificial chromosome (BAC) DNA]. A rapid and baselineresolved separation of PmeI digested Rice BAC DNA fragments was achieved at a speed of ∼25 min per run.

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EXPERIMENTAL SECTION Reagents and Materials. Lambda Mix Marker 19 was purchased from Fermentas Life Sciences Inc. (Glen Burnie, MD). Lambda DNA Mono Cut mix and restriction enzyme PmeI were obtained from New England Biolabs Inc. (Beverly, MA). Tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), boric acid, and sodium hydroxide were purchased from Fisher Scientific (Fisher, PA). YOYO-1 was obtained from Molecular Probes (Eugene, OR). Large amount plasmid preparation kits were purchased from Aidlab (Beijing, China). Biowest regular agarose G-10 was obtained from Gene Company (Hongkong, China). Fused-silica capillaries (20.0 μm, and 5.0 μm diameter) were supplied by Polymicro Technologies (Phoenix, AZ). Fused-silica capillary (1.5 μm diameter) was specially produced by Polymicro Technologies. 1 × TE buffer (10 mM TE buffer: 10 mM TrisHCl, 1 mM Na2EDTA, pH 8.0) was prepared from a 10-fold concentrated stock (100 mM Tris base and 10 mM Na2EDTA; pH adjusted to 8 with a concentrated HCl) by dilution with sterilized DDI water. The stock solution was autoclaved and then stored at 4 °C. All solutions were prepared using ultrapure water (Nanopure ultrapure water system, Barnstead, Dubuque, IA) and filtered through a 0.22 μm filter (Derian, Shanghai, China), and vacuum degassed before use. Narrow, Bare Open Capillary-Based Separation Devices. Figure S1 of the Supporting Information shows a schematic representation of the apparatus used in our earlier work.34 Briefly, a septum-sealed pressure chamber device was constructed in-house. A 600 μL microcentrifuge tube, used as the solution vial, was mounted in the pressure chamber. The sampling end of the capillary was inserted through the septum into the solution vial inside the pressure chamber. Pressureregulated helium gas was introduced to the pressure chamber to drive the solution in the vial through the capillary. At an appropriate location near the end of the capillary (∼5 cm from the terminus), a detection window was formed by removing off a small segment of the polymer coating. The detection end of the capillary was held firmly in place by an in-house built holder attached to an XYZ micromanipulator (Newport M-460AXYZ) (Newport Corporation, Irvine, CA) to align the detection window with the optical system. The fluorescence measurement was performed on a home-built confocal laserinduced fluorescence detector we described previously.38 Preparation of Standard DNA Samples. The doublestranded DNA (dsDNA) samples were obtained by mixing the

Q=

πR′2 l t

(1)

where R′ = 10 μm, l is the distance the meniscus moved, and t is the time of the test. 730

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Effect of Capillary Length on Retention Factor. DNA separations were conducted in a narrow, bare open capillary under a pressure-driven flow in free solution (see Figure S1 of the Supporting Information). Capillary length is a key parameter that influences the separation efficiency. The effect of capillary length on separation performance was tested with a constant pressure gradient. Figure 2a displays the separation

RESULTS AND DISCUSSION Figure 1 presents a schematic illustration of an HDC separation of DNA molecules. DNA molecules inside a capillary with a

Figure 1. Schematic diagram of DNA separation in a narrow, bare open capillary. The chromatogram is the separation of the Lambda DNA Mono Cut Mix.

radius of R are carried by a pressure-driven flow that is laminar with the familiar Poiseuille pattern of streamline velocities, highest in the middle and diminishing toward the wall. When two DNA molecules with radii of r1 and r2 are inside the capillary, their movement is affected both by the slower velocities near the wall and the faster velocities near the center. Thus, larger molecules [having a smaller effective capillary diameter of 2(R − r2)] are more affected by the faster flow near the center of the capillary than the smaller molecules [having a larger effective capillary diameter of 2(R − r1)] and therefore travel faster than the smaller ones. Retention Factor. The rate of transport of DNA fragments through the narrow capillaries is calculated from the ratio of capillary length to the average retention time of DNA fragments. The retention factor Rf, the rate of transport of the DNA fragments (QD) relative to the eluant (Q0), can be expressed by39 Rf =

QD Q0

=

t0 tD

Figure 2. Separation of Lambda DNA Mono Cut Mix. (a) Effect of capillary length on resolution by narrow, bare open capillary chromatography. The separations were carried out in 5 μm i.d. capillary under 1 psi/cm pressure gradient with different capillary lengths: (i) 500 cm total long and 495 cm effective, (ii) 300 cm total long and 295 cm effective, (iii) 200 cm total long and 195 cm effective, and (iv) 50 cm total long and 45 cm effective. Separation pressures were as follows: (i) 500, (ii) 300, (iii) 200, and (iv) 50 psi, respectively. Sample injection conditions: (i) 9 s at 80 psi, (ii) 5 s at 60 psi, (iii) 4 s at 40 psi, and (iv) 4 s at 12 psi. Eluent was 10 mM TE (pH = 8.0). (b) PFGE separation of 0.5 μg of Lambda Mono Cut Mix. 1% agarose gel, 0.5X TBE, 6 V/cm, 15 °C for 20 h. Switch times ramped from 0.5 to 1.5 s. (Reprinted from www.neb.com (2013) with permission from New England Biolabs.) (c) The fitting result of trace (i) in (a). Black squares are experiment data; the red line is the fitting.

(2)

where t0 and tD demonstrate the elution times of the eluant and DNA fragments. The elution time of the eluant, t0, was calculated from the measured eluant flow rate (see the Experimental Section), the diameter, and the effective length of the capillary. In HDC, the retention factor is always greater than one because DNA fragments travel through the capillary faster than the average flow of the eluant.

results of Lambda DNA Mono Cut Mix containing 10 DNA fragments (1.503, 10.086, 15.004, 17.053, 23.994, 24.508, 29.946, 33.498, 38.416, and 48.502 kbp) with the pressure gradient of 1 psi/cm. The DNA molecules eluted out strictly, according to their sizes with larger DNA fragments moving faster than the smaller ones. Under these conditions, the resolution between the DNA molecules increased with 731

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Table 1. Calculated the Number of Theoretical Plates for the Peaks in the Trace i of Figure 2a DNA length (kbp) N H (μm)

48.502 3.6 × 105 13.9

38.416 2.7 × 105 18.1

33.498 2.6 × 105 18.9

29.946 4.3 × 105 11.6

24.508 1.4 × 105 34.6

increasing capillary length, as expected. From trace (i), it appears that peak shapes are quite symmetrical. Figure 2b shows the results obtained from PFGE provided by New England Biolabs Inc. on its Web site. The separation [shown in trace (i)] took about 2 h, while the PFGE separation took about 20 h. As indicated by the results in traces (ii and iii) of Figure 2a, the Lambda DNA Mono Cut Mix could be baselineresolved in less than one hour. It is notable that our method presents greater efficiency than the PFGE method. Separation efficiencies are usually represented by the number of theoretical plates. We calculated the number of theoretical plates for all the peaks in the bottom trace of Figure 2a, and the results are listed in Table 1. It reached 3.6 × 105 plates for 48.5 kbp and 3.7 × 105 plates for 1.5 kbp, presenting the highest efficiencies ever reported for separations of large DNA molecules, using a chromatographic technique. Often, the height equivalent of a theoretical plate (HETP or H) is used to express the column efficiency for a particular analyte. Using the effective column length (500 cm) and the plate numbers from Figure 2a, we computed the H values, and these data are also presented in Table 1. These H values are in good agreement with theoretically predicted results.40 In open tubular HDC, the height equivalent of a theoretical plate can be evaluated by the Golay equation,41,42

H=

2Dm ud 2 + u 96Dm

Rf = 1 + 2

⎛ r ⎞2 r − C⎜ ⎟ ⎝R⎠ R

(3)

(4)

where r and R are the radii of the DNA fragments and the capillary. In accordance with literature,40 a polymer molecule can be treated as a particle having an effective hydrodynamic radius of RHD, and RHD = k × Lυ

17.053 1.6 × 105 30.6

15.004 2.0 × 105 25.0

⎛ k × Lυ ⎞2 k × Lυ ⎟ − C⎜ ⎝ R ⎠ R

10.086 1.3 × 105 39.5

1.503 3.7 × 105 13.2

(6)

The experimental data and result fitting from Figure 2a trace (i) are shown in Figure 2c. The retention factor changed proportionally with L. When we modified eq 6 by adding a term qL (where q is a constant) to the right side of the equation, an excellent correlation coefficient (r2 = 0.9998) was obtained, which indicated that the separation mechanism is hydrodynamic chromatography, and the modified equation worked satisfactorily. Effect of Eluant Average Velocity on Retention Factor. Eluant velocity (or elution pressure) is another important parameter for the separation. The effect of elution pressure on the retention factor of each fragment of Lambda DNA Mono Cut Mix in the same length capillaries was investigated and illustrated in Figure S2 of the Supporting Information. These results show that the retention factor clearly increases with increasing DNA length due to larger DNA fragments having smaller effective internal capillary diameters, which suggests a foundation of a chromatographic size separation. The retention factor of large DNA fragments depends on the elution pressure (the eluant velocity), while the retention factor of small DNA fragment (1.5 kbp) remains nearly constant and shows independence with the elution pressure. For large DNA fragments, the retention factor increases with increasing elution pressure. At higher eluant velocities, the increasing retention factor is more pronounced than at lower eluant velocities, which could be caused from the tubular pinch effect.44 The slope of retention factor vs elution pressure increases when the DNA length increases, and then slightly decreases, which may be due to the deformation of larger DNA fragments in the same capillary. Effect of Eluant Concentration on Retention Factor. The separation results of Lambda DNA Mono Cut Mix in free solutions with different TE concentrations are presented in Figure S3 of the Supporting Information. It is apparent that the resolution of separated DNA fragments improved with the increase of eluant concentration, and the effect of eluant concentration on retention factor is illustrated in Figure 3. The retention factor increases with increasing eluant concentration. This phenomenon is the reverse of that reported for the separation of latex particles,39 which could be caused by the association of more counterions (e.g., Na+) with DNA molecules at higher eluant concentrations. This could also explain that the extent of the increase in the retention factor with increasing of eluant concentration is higher for larger DNA fragments than for smaller ones. Effect of Separation Temperature on Retention Factor. The separation could be accelerated by increasing the separation temperature, which was controlled by inserting the capillary through a silicon tube connected to a circulating water bath. The separation results areas of Lambda DNA Mono Cut Mix in free solutions at different separation temperatures are presented in Figure 4a. An increase in temperature results in a decrease in the viscosity of the eluant, which enhanced the separation rate. It was seen that the speed of separation nearly

where Dm is the diffusion coefficient, u is the linear velocity of the eluent, and d is the column diameter. For trace (i) in Figure 2a, d = 5.0 × 10−6 m and u = 5.21 × 10−4 m/s. The values of Dm were then calculated using eq 4 and presented in Table 1. We noticed that m1, and RS is the resolution between two different sizes of DNA R S = 1.18

t 2 − t1 w0.5,2 + w0.5,1

(8)

where t1 and t2 are the retention times, and w0.5,1 and w0.5,2 are the widths at half height. For example, the resolution (RS) between peaks of 48.502 and 38.416 kbp in Figure 2a trace (i) is 2.7, and the corresponding percentage resolution (Rp) is 9.0%. That is, two DNA fragments with a 9.0% size difference can be resolved at Rs = 1. A resolution (RS) of 2.9 between peaks of 17.053 and 15.004 kbp and the corresponding Rp of 4.6% mean the ability to resolve a 4.6% difference in molecular weight. Application for Separation of Sac II Digested Arabidospois BAC DNA. To demonstrate the practical applicability of this technique, we separated Sac II digested DNA prepared from the Arabidospois T6B20 BAC clone, shown in Figure 5. T6B20 BAC DNA contains a 104.9 kbp genomic DNA insert from Arabidopsis chromosome II and a 7.5 kbp pBeloBAC11 vector backbone. Digestion of T6B20 BAC DNA with Sac II restriction endonuclease is expected to produce DNA fragments of 53.5, 46.8, and 12.1 kbp sizes. Separation of restriction digestion products on agarose gel showed only two bands, including one slow migrating band with an apparent size of approximately 50 kbp and a faster migrating band of about 12.1 kbp. It is likely that the slow DNA band includes both the 53.5 and 46.8 kbp fragments. In order to resolve the two large fragments, the restriction digestion product was allowed to run on the agarose gel for 24 h. It was still not possible to separate the two expected bands, shown in 734

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performance than slab-gel electrophoresis for large DNA and more rapid separation than PFGE for a wider size range for fragment sizes.

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ASSOCIATED CONTENT

S Supporting Information *

Instrument setup and additional figures as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ⊥

Department of Biological Sciences, University of North Texas, Denton, TX 76203, United States.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 20935002, 21075006, and 21005005), Beijing Natural Science Foundation (Grant 2112003), and the Program for New Century Excellent Talents in University (Grant NCET-12-0603). We thank Arizona Genomics Institute and Prof. Yonghong Wang of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences for providing the Rice BAC plasmid DNA; Prof. Yuxin Hu and Baoshuan Shang of the Institute of Botany of the Chinese Academy of Sciences for technical assistance in BAC DNA preparation and restriction digestion; Yaxin Zhu of the Institute of Microbiology of the Chinese Academy of Sciences for technical assistance in PFGE separations; Prof. Purnendu K. Dasgupta of the University of Texas at Arlington for discussions on this manuscript.

Figure 6. Separation results of PmeI digested Rice OSJNBa0088H09 BAC DNA. (a) Narrow, bare open capillary separation results. The separations were carried out in 400 cm long and 5 μm i.d. capillary at 40 °C under 900 psi. The eluent contained 10 mM TE. The sample was injected at 100 psi for 15 s. Trace A shows the results of 60 ng/μL Lambda DNA Mono Cut Mix, and trace B exhibits the result from 50 ng/μL PmeI digested Rice BAC DNA. (b) PFGE separation results: Rice BAC DNA was separated in 1.0% agarose gel in 0.5× TBE buffer at 3 V/cm and 14 °C for 15 h with a ramped pulse time from 0.5 to 1.5 s. Left lane is loaded with 500 ng of the Lamda DNA Mono Cut Mix. Right lane is loaded with 1.2 μg PmeI digested Rice BAC DNA. (c) Separation results of 50 ng/μL PmeI digested Rice BAC DNA. Separations were carried out in 250 cm long and 5 μm i.d. capillary at 40 °C under 900 psi. The eluent contained 10 mM TE. The sample was injected at 100 psi for 8 s.

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REFERENCES

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CONLCUSIONS We have experimented with HDC for separating specifically large DNA up to ∼120 kbp using bare, narrow open capillaries. Lambda DNA Mono Cut Mix could be well-resolved in about 2 h, compared to more than 20 h using PFGE at comparable resolutions. The resolutions were generally less than 9.0% for the all DNA fragments with efficiencies of >105 plates, the highest efficiencies ever obtained by a chromatographic technique for large DNA fragments in free solution. We also have analyzed the performance and evaluated the basic parameters of HDC; the results are in good agreement with theoretically predicted values. On the basis of these data, we can conclude that narrow, bare open capillary HDC is an excellent alternative to PFGE for separation of DNA fragments from a few hundred base pairs to a few hundred kilo base pairs. The feasibility for real-world sample separation was demonstrated, showing that this technique has superior separation 735

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Analytical Chemistry

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dx.doi.org/10.1021/ac403190a | Anal. Chem. 2014, 86, 729−736