Anal. Chem. 2001, 73, 6053-6056
Separation of 100-Kilobase DNA Molecules in 10 Seconds Olgica Bakajin,†,| Thomas A. J. Duke,‡ Jonas Tegenfeldt,† Chia-Fu Chou,† Shirley S. Chan,† Robert H. Austin,*,† and Edward C. Cox§
Physics Department, Princeton University, Princeton, New Jersey 08544, Cavendish Laboratory, Madingley Road, Cambridge CB3 0HE, U.K., and Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Long double-stranded DNA molecules were separated in microfabricated hexagonal arrays in less than 1 min, several orders of magnitude faster than by using conventional technology. DNA samples were first concentrated at the entrance to the array in a thin band by entropic focusing. They were then separated by pulsed field electrophoresis. T4 (168.9 kbp) and λ (48.5 kbp) DNAs could be resolved into two clearly separated bands in ∼10 s in these experiments. This corresponds to a mass resolution of 6% in 11 min in a 1-cm-long array. DNA molecules of different lengths are usually separated by agarose gel electrophoresis. This method, however, loses its efficiency for molecules longer than ∼20 kbp. For molecules much larger than this, pulsed field gel electrophoresis1,2 is commonly used. The pulsed field gels themselves are also limited, both with respect to speed (runs take many hours to many days) and molecular weight range, with an upper limit of ∼10 Mbp.3,4 Transverse pulsed field electrophoresis in hexagonal arrays uses an array of 2-µm pillars with 2-µm spacings arranged in a hexagonal lattice and takes advantage of the DNA elongation that occurs in microfabricated arrays.5 Application of a pulsed field along alternating axes of the array, separated by 120°, causes net motion of the DNA molecules along the bisector of the axes, with average migration speeds that depend on their length (Figure 1). A useful separation device, in addition to using an effective separation mechanism, must also collect and launch molecules in a narrow zone, since initial zone broadening destroys resolving power. In our device, the DNA was entropically trapped and released as a band using the principle described by Han and Craighead.6 We used an entropic barrier placed in the path of * Corresponding author: (e-mail)
[email protected]). † Physics Department, Princeton University. ‡ Cavendish Laboratory. § Department of Molecular Biology, Princeton University. | BioSecurity Support Laboratory, Lawrence Livermore Laboratory, Livermore, CA. (1) Schwartx, D. C.; Cantor, C. R. Cell 1984, 37, 67-75. (2) Carle, G. F.; Frank, M.; Olson, M. V. Science 1986, 232, 65-68. (3) Cox, E. C.; Vocke, C. D.; Walter, S.; Gregg, K. Y.; Bain, E. S. Proc. Natl. Sci. U.S.A. 1990, 87, 8247-8251. (4) Orbach, M. J.; Vollrath, D.; Davis, R. W.; Yanofsky, C. Mol. Cell. Biol. 1988, 8 (4), 1469-1473. (5) Volkmuth, W. D.; Austin, R. H. Nature 1992, 358, 600-602. (6) Han, J.; Craighead, H. G. Science 2000, 288, 1026-1029. 10.1021/ac015527o CCC: $20.00 Published on Web 11/02/2001
© 2001 American Chemical Society
Figure 1. Microscopic view of the hexagonal array. Pillars 2 µm wide were used in these experiments. Cartoon DNA molecules are drawn to illustrate the motion of individual molecules of different lengths. Each period T consists of two pulses aligned along the channels created by the posts in the array, giving a net angle between the two field directions of 120°. Shorter molecules move farther in the array because once they have reoriented along the axis of the field they move in an unhindered straight line for the duration of the pulse. Longer molecules, on the other hand, spend most of the pulse period retracing their paths. In the example shown here, the longer of the two will never advance.
the DNA near the entrance to the array (Figure 2). There is a small gap between the barrier top and the cover slip that seals the array. The gap between the top of the barrier and the cover slip was smaller than the radius of gyration of the DNA molecules to be fractionated (150 nm in these experiments). When very low dc fields are applied to move DNA molecules into the array, the molecules do not have enough applied force applied to them to squeeze through the gap, and hence they get trapped (Figure 2B). Under higher fields, they get stretched and move through the gap with mobilities that are independent of molecular length. In our device, molecules were first transported into the array through the gap using a high electric field. They were then concentrated against the barrier by applying a low electric field oriented in the opposite direction. Concentrated molecules were launched into the array by reversing the field direction. The alternating fields were not applied directly at the entropic barrier but rather were applied only after the DNA band had been moved several millimeters into the array. This was done to avoid the field curvature seen at the corners of the array and ensure that the fields were uniform in the directions necessary for predictable fractionation. Analytical Chemistry, Vol. 73, No. 24, December 15, 2001 6053
Figure 3. Video images of DNA separation. Video clips of λ and T4 DNA pulsed at 244 V and with period T ) 1 s after release from the entropic trap.
Figure 2. Device configuration. (A) Sketch of the device. The diameter of a typical device was 1-3 cm. The pulsed field was applied through two pairs of external electrodes (C-D and E-F). The remaining electrode pair (A-B) was used for entropic trapping. The electrodes were insulated from each other by six silicone structures (lozenge shaped in panel A). (B) Cross section of the device showing entropic trapping. The arrows point in the direction of DNA motion, while their lengths correspond to the strength of the applied field. (BI) The beginning of DNA transport across the barrier using a high electric field. (BII) The concentration and cleanup step where the molecules are forced back against the barrier at low field before they are launched into the array.
EXPERIMENTAL SECTION The devices were made of quartz using standard photolithography and reactive ion etching techniques. They contained an array of pillars oriented in a hexagonal lattice whose height was 2 µm. The arrays were sealed using glass cover slips with a spunon thin-layer silicone elastomer7 (RTV615A and RTV615B, GE Silicones, Waterford, NY). The silicone elastomer surface was treated for 1 min in an oxygen plasma to make the silicone hydrophilic, necessary for wetting of the sealed device. The devices were shaped as hexagons 3 cm in diameter (Figure 2A) to allow easy application of electric fields oriented at 120°.8 They were mounted on a plastic holder that contained the outside electrodes. Two pairs of outside electrodes were used to apply pulsed fields (C-D, E-F; Figure 2) while the remaining pair was used for entropic trapping (A-B). The DNA fluoresence stain TOTO-1 (Molecular Probes) was used at 1 µg/mL concentrations to stain T4 and λ DNA molecules which were loaded into the arrays in concentrations of 15 and 5 µg/mL, respectively, and observed by epifluorescence using the 488-nm line of an Ar/Kr laser. The 0.5× TBE electrophoresis buffer (45 mM Tris/borate, 1 mM EDTA, pH 8.0) contained 0.1% POP-6 (Perkin-Elmer Biosystems) to reduce electroendosmosis and 0.1 M DTT to reduce bleaching. (7) Duffy, D. C.; McDonal, J. C.; Schueller, O. J.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (8) Chu, G.; Vollrath, D.; Davis, R. W. Science 1986, 234, 1582-1585.
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Figure 4. DNA position as a function of time. The position and speeds of the band peaks of λ (squares), T4 (circles), and the mixed band (diamonds) vs time. From t1 until t2, the pulsing parameters were 100 V and T ) 1 s. The peak of the mixed band moved at 2.3 µm/s. At time t2, the voltage was increased to 244 V. The two bands were cleanly resolved in ∼10 s. T4 DNA moved at 5.6 µm/s, and λ DNA moved at 14.9 µm/s. Speeds were obtained by fitting the straight lines shown through the band centroid positions.
EXPERIMENTAL RESULTS T4 (168.9 kbp) and λ (48.5 kbp) DNA were separated in a very short time with high resolution. The mixture (see Experimental Section) was resolved into two bands in 10 s (Figures 3 and 4). The position of the peaks is plotted versus time in Figure 4. From t1 ) -10 s to t2 ) 0 s, the applied voltage was 100 V and the pulse period T was 1 s. Under these conditions, the bands separated by less than the bandwidth. At time t2, the voltage was increased to 244 V, keeping the period the same. The two bands were then resolved in ∼10 s. Figure 5 confirms that under our pulsed field conditions the shorter molecules move faster than the longer molecules. The widths of the bands after 11 min were approximately the same for both DNAs (fwhm was ∼200 µm). For a diffusion constant D ∼ 1 µm2/s, the expected diffusional broadening (∼ [2Dt]1/2 due to self-diffusion in t ) 11 min is ∼30 µm, while the observed broadening is 100 µm. This dispersive broadening possibly occurs because individual molecules of equal lengths get stretched by different amounts, or some times not at all, when encountering posts. The location of the bands after 11 min was consistent with the band separation derived from the microscopic migration velocities, ∆x ) (Vλ - VT4)t ) 6100 µm. The band
time to reorient completely during a single pulse repeatedly retrace the same path, and since they make no progress, their velocity is effectively zero. The reorientation time tor is to first order simply L/µoE, or more empirically given by
tor ) c1L/(µoE)
(2)
where c1 is a parameter that takes into account the fact that the DNA molecules are not fully aligned. Roughly, to fractionate molecules of length L, the pulse period T should be set to tor. Thus, the upper limit of separation, L*, is proportional to the pulse time T and to the speed µoE of a molecule along a free channel:
L* ) (T/2)(µ0E/c1) Figure 5. Single-molecule images. Images of single molecules taken at 11 min after the start of pulsing. These images were gathered 10 (left) and 4 mm (right) from the entropic barrier. In the images at the top, the field was turned off and the molecules were at thermal equilibrium, while in the bottom panels the separated molecules were elongated under pulsed field conditions. Note that the radius of gyration of the relaxed molecules in the top panels clearly establishes that the faster moving species is λ DNA.
capacity9 nC in this experiment is estimated to be ∆x/[1.5[σ(λ) + σ(T4)] ) 20; i.e., 20 bands could be resolved in the 50-170 kbp range under the conditions used here. Since the separation is approximately linear in molecular weight (see below), this means that molecules which differ by 6 kbp can in principle be distinguished. Observation of the microscopic dynamics confirms that the separation is a consequence of “switchback” motion of the DNA molecules for opposing field directions greater than 90°, as reported in preliminary investigations10,11 and illustrated in Figure 1. When the field direction is switched, each molecule moves off in the new field direction, led by the end that was previously trailing. As a result, the molecules retrace part of the path they have traveled. Since longer molecules backtrack further than shorter molecules, the rate of advance along the bisector of the field is slower. The overall speed of migration in a pulsed hexagonal array is length-dependent. Under the simplifying assumption that the molecules remain uniformly stretched during this motion, the net velocity VL of molecules of length L in a pulsed field array can be described by the simple equation10
VL ) µoE cos(θ/2)[1 - L/L*]
(1)
where the angle θ between transverse fields in our case is 120° and µo is the continuous-field mobility, which is independent of length, and L* is a critical cutoff parameter. The critical cutoff length L* arises from that fact that molecules which do not have (9) Giddings, J. C. Unified Separation Science; John Wiley & Sons: New York, 1991; pp 101-106. (10) Duke, T. A. J.; Austin, R. H.; Cox, E. C.; Chan, S. S. Electrophoresis 1996, 17, 1075-1079. (11) Bakajin, O.; Duke, T. A.; Chou, C. F.; Tegenfeldt, J.; Chan, S. S.; Austin, R. H.; Cox, E. C. Third International Biophysics Symposium Proceedings, AIP Conference Proceedings 1998, 487, 243-248.
(3)
It is a strength of the analytical nature of the dynamics of DNA molecules in synthetic arrays that L* and T can be predicted. Note that we can obtain expressions for µoE and L* from our experimental data at 244 V with a pulse period T of 1 s for T4 and λ DNA. Some algebra gives
µoE )
LT4Vλ - LλVT4 (LT4 - Lλ) cos(θ/2)
L* )
LT4Vλ - LλVT4 (VT4 - Vλ)
(4)
(5)
These expressions can then be used to check the actual parameters used in the experiment. We can assume (somewhat incorrectly) that the lengths of the DNA in the above expression are the fully stretched lengths taking into account that one Kuhn length b contains 300 base pairs and is 130 nm long for TOTO1-stained DNA,12 giving LT4 ) 168.9 kbp ) 73 µm and Lλ ) 48.5 kbp ) 21 µm. The measured values of the retarded velocities are VT4 ) 5.6 µm/s and Vλ ) 14.9 µm/s; from these parameters we get µoE ) 37 µm/s and L* ) 104 µm. The pulse periods T that should be used at L* are then roughly tor ) 3 s. Thus, pulse periods on the order of 1 s in this particular protocol are appropriate. We can check the consistency of the experimental data at the two field strengths. Since the migration velocity in a continuous field is proportional to the applied voltage, we expect that, at 100 V, µ0 E ) 15 µm/s. Equation 3 then predicts that L* ) 43 µm. Equation 1 predicts that λ DNA should move at velocity Vλ ) 3.9 µm/s, which is what we observe. Since the T4 DNA is longer than L* at this field strength, the theory predicts that it will not move, but in practice, it advances at the low speed of 2 µm/s. This migration occurs because the backtracking motion is not ideal. We observe that the molecules do not remain uniformly extended, but usually get stretched by the field and subsequently relax during each pulse. Because of this inherent stochasticity, even the longest molecules do not retrace the same path indefinitely. Our measurements yield c1 ) 0.18 (eq 3). Duke et al.10 calculated the value c1 ) 1.39, assuming that molecules are extended to their full contour length at all times. If the molecules (12) Bakajin, O. B.; Duke, T. A.; Chou, C. F.; Chan, S. S.; Austin, R. H.; Cox, E. C. Phys. Rev. Lett. 1998, 80, 2737-2740.
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are stretched to only a fraction of their length, we would expect the value of c1 to be proportionately reduced, because the reorientation time would be faster. The lower value of c1 that we measure is therefore consistent with our observations that, at the field strengths used, the molecules are rarely extended to more than 30-40% of their full contour length. DISCUSSION Long DNA molecules in agarose gels and other polymer matrixes get hooked on many gel fibers simultaneously, exhibiting complex motion, and confounding theory and experiment alike. The simpler array dynamics allows better prediction of pulsed field parameters for a given range of molecular sizes, and a relatively simple theory describes motion. This analytical nature of the motion is a real advantage of the technique since it may allow us to separate unlabeled molecules Separation in the hexagonal arrays will be even faster when higher electric fields with shorter periods are applied. In our experiments, we were limited to relatively low electric fields and long periods because we wished to record single-molecule images. Higher fields would cause greater molecular extension, which would enhance the regularity of the “switchback” motion and improve the discrimination between molecules of different size. Reduction of the depth of the device is also expected to increase the extension of the molecules.12 We can compare these results with those of others. Pulsed field capillary gel electrophoresis13-14 achieves fast separation, but this method is severely limited by the tendency of high molecular weight DNA to form supramolecular complexes that interfere with separation. Chou et al.15 have proposed a single-molecule sizing device in which molecules in the 2-200 kbp range are sized one at a time. This method, however, cannot be used to separate many thousands of molecules simultaneously. Other methods, such as recently developed arrays that separate molecules based on their diffusion coefficients,16 suffer from rapidly deteriorating resolution as the molecules get bigger. Han and Craighead6 have shown that entropic trapping can be used to separate molecules in reverse (13) Kim, Y.; Morris, M. D. Anal. Chem. 1995, 67, 784-786. (14) Magnusdottir, S.; Isambert, H.; Heller, C.; Viovy, J. L. Biopolymers 1999, 49, 385-401. (15) Chou, H. P. Spence, C.; Scherer, A.; Quake, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11-13. (16) Chou, C. F.; Bakajin, O. B.; Turner, S. W.; Duke, T. A. J.; Chan, S. S.; Cox, E. C.; Craighead, H. G.; Austin, R. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13762-13765.
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order, with the largest molecules moving the fastest. This method, although faster than conventional methods, is still at least 1 order of magnitude slower than separation in hexagonal arrays using quantitatively understood pulsed field parameters that we report here. The separation principle that we demonstrate here is neither limited to DNA molecules of a particular size nor does it require the molecules to be fluorescently labeled. By appropriately adjusting the applied electric field, the pulse time, and array parameters such as the pillar size or spacing, the technique could be extended to the separation of polymers of all lengths. Because the motion of the molecules in hexagonal arrays is predictable, it should be possible to operate the device “blind”, without the addition of dye stains which can contaminate subsequent processes such as polymerase chain reactions. Alternatively, the dye can be removed by standard techniques using ion-exchange resins. In the future, it should be feasible to dispense with fluorescence-based detection methods and detect the DNA molecules electronically using nanosensors constructed on the chip.17 CONCLUSION We have shown that fast DNA separation with high resolution can be achieved in hexagonal arrays. Our results represent an increase in separation speed of many orders of magnitude and also show that array technology can exhibit high resolution. Since these arrays do not require a polymer matrix as a fractionation medium, they are suitable for an integrated bioanalysis system18 and are easy to describe mathematically. In the future, it will be possible to integrate this device with whole cell preparations, so that Mbp-sized DNA molecules can be karyotyped directly and in a few seconds. ACKNOWLEDGMENT The authors thank J. Han for his helpful suggestions and early contributions to this work. This research was supported by NIH Grants GM55453 and HG01506. O.B. acknowledges support by a Fellowship from the Program in Mathematics and Molecular Biology at the Florida State University, with funding from the Burroughs Wellcome Fund Interfaces Program. T.A.J.D. acknowledges support from BBSRC Grant E08580. AC015527O (17) Duan, X.; Huang, Y.; Cui, Y.; Wang, J.; Lieber, C. M. Nature 2001, 409, 66-69. (18) Manz, A.; Graber, N.; Widmer, H. M. Sens. Acutators, B 1990, 1, 244-248.
