Automation and Integration of Multiplexed On-Line Sample

Anal. Chem. 1998, 70, 4044-4053

Automation and Integration of Multiplexed On-Line Sample Preparation with Capillary Electrophoresis for High-Throughput DNA Sequencing Hongdong Tan and Edward S. Yeung*

Ames LaboratorysUSDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011

An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at ∼60 °C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at ∼70 °C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing. From its inception, the Human Genome Project1 has called upon developing technologies for cost-effective, high-speed, and high-throughput DNA sequencing because of the inadequate overall efficiency of traditional slab gel electrophoresis. Alternative technologies such as capillary array electrophoresis,2 microchannel array electrophoresis,3 sequencing by hybridization,4 singlemolecule sequencing,5 and sequencing by mass spectroscopy6 have been explored extensively. Intensive research efforts have also been carried out in improving individual technologies from (1) (a) Joint U.S. Department of Energy and U.S. Department of Health and Human Services Report DOE/ER-0452P. Understanding Our Genetic InheritancesThe U.S. Human Genome Project: The First Five Years, Washington, DC, April 1990. (b) Collins, F., Galas, D. Science 1993, 262, 43-46. (2) (a) Huang, X. C.; Quasada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967-972. (b) Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424-1431. (c) Dovichi, N. J.; Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R. Anal. Chem. 1991, 63, 2835-2841. (d) Chen, D. Y.; Swerdlow, H. P.; Harke, H. R.; Zhang, J. Z.; Dovichi, N. J. Chromatogr. 1991, 559, 237-246. (e) Mathies, R. A.; Huang, X. C. Nature 1992, 359, 167-169. (f) Takahashi, S.; Murakami, K.; Anazawa, T.; Kambara, H. Anal. Chem. 1994, 66, 10211026.

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template preparation, reaction chemistry, purification, sample injection, separation, detection, and data analysis. However, it is the integration and automation of all the individual technologies that is critical to success of Human Genome Project. Capillary electrophoresis (CE) is an attractive approach to replace conventional slab gel electrophoresis due to advantages including high migration speed, high separation efficiency, small sample requirement, and suitability for automation. System integration of CE to robotic arms and conveyer belts,7 although workable, suffers from reliability and incompatibility issues because of the many moving parts at the robotic end and the small volumes at the separation and detection end. On the other hand, (3) (a) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348-11352. (b) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (c) Raymond, D. E.; Manz, A.; Widmer, M. H. Anal. Chem. 1994, 66, 2858-2865. (d) Balch, J. W.; Davidson, C.; Gingrich, J.; Sharaf, M.; Brewer, L.; Koo, J. W.; Smith, D.; Albin, M.; Carrano, A. Genome Sequencing and Analysis Conference VI, September 17-21, 1994, Hilton Head, SC, Abstract C2. (4) (a) Drmanac, R.; Crkvenjakov, R. Yugoslav Patent Application 570, 1987. (b) Southern, E. United Kingdom Patent Application GB 8810400, 1988. (c) Bains, W.; Smith, G. C. J. Theor. Biol. 1988, 135, 303-307. (d) Lysov, Y. P.; Floretiev, V. L.; Khorlyn, A. A.; Krhapko, K. R.; Shick, V. V.; Mirzabekov, A. D. Dokl. Acad. Nauk SSSR 1988, 303, 1508-1511. (e) Drmanac, R.; Labat, I.; Brukner, I.; Crkvenjakov, R. Genomics 1989, 4, 114-128. (f) Khrapko, K. R.; Lysov, Y. P.; Khorlyn, A. A.; Shick, V. V.; Florentiev, V. L.; Mirzabekov, A. D. FEBS Lett. 1989, 256, 118-122. (g) Macevicz, S. C. International Patent Application PC US89 04741, 1989. (h) Drmanac, R.; Drmanac, S.; Labat, I.; Crkvenjakov, R.; Vicentic, A.; Gemmell, A. Electrophoresis 1992, 13, 566-573. (5) (a) Davis, L. M.; Fairfield, F. R.; Harger, C. A.; Jett, J. H.; Keller, R. A.; Hahn, J. H.; Krakowski, L. A.; Marrone, B. L.; Martin, J. C.; Butter, H. L.; Ratliff, R. L.; Shera, E. B.; Simposon, D. J.; Soper, S. A. Genet. Anal. 1991, 8, 1-7. (b) Goodwin, P. M.; Cai, H.; Jett, J. H.; Ishaug-Riley, S. L.; Machara, N. P.; Semin, D. J.; Van Orden, A.; Keller, R. A. Nucleosides Nucleotides 1997, 16, 543-547. (6) (a) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946. (b) Xu, L.; Bian, N.; Wang, Z.; AbdelBaky, S.; Pillai, S.; Magiera, D.; Murugaiah, V.; Giese, R. W.; Wang, P.; O’Keefle, T.; Abuskaman, H.; Kutney, L.; Church, G.; Carson, S.; Smith, D.; Park, M.; Wronka, J.; Laukien, F. Anal. Chem. 1997, 69, 3597-3602. (c) Smith, L. M. Science 1993, 262, 530-532. (d) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLaffery, F. W. J. Am. Chem. Soc. 1994, 116, 4893-4897. (e) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLaffery, F. W. Anal. Chem. 1994, 66, 2809-2815. (f) Wu, K. J.; Shalter, T. A.; Becker, C. H. Anal. Chem. 1994, 66, 1637-1645. (g) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, 215A-229A. (7) (a) Mardis, E. R.; Roe, B. A. BioTechniques 1989, 7, 840-850. (b) Wilson, R. K.; Yuan, A. S.; Clark, S. M.; Spence, C.; Arakelian, P.; Hood, L. E. BioTechniques 1988, 6, 776-777. (c) Zimmermann, J.; Voss, H.; Schwager, C.; Stegemann, J.; Angsorge, W. FEBS Lett. 1988, 233, 432-436. (d) DeAngelis, M. M.; Wang, D. G.; Gawkins, T. L. Nucleic Acids Res. 1995, 23, 4742-4743. (e) http://hgighub.lbl.gov/esd/DNAPrep/Titlepage.html. S0003-2700(98)00406-5 CCC: $15.00

© 1998 American Chemical Society Published on Web 09/03/1998

on-line integrated microfluidics is inherently more compatible with the capillary/microchannel formats. Based on this approach, DNA restriction digestion,9 polymerase chain reaction,10 and cycle sequencing reaction11 have all been interfaced with capillary/ microchannel electrophoresis for sizing. Although these studies successfully coupled sample preparation with separation and detection in a single channel, multiplexing of the same schemes has not been demonstrated. A high degree of multiplexing of parallel on-line runs must be implemented in large-scale sequencing no matter how fast, small, and low cost the devices are. We note that strategies that work best when small numbers of samples with few time constraints are analyzed may not be transferable to a large-scale project. It will therefore be necessary to reoptimize the performance of each of the critical steps in the operation to achieve a workable compromise for large-scale applications. Previously, we showed that an on-line integrated microfluidic system from dye-terminator sequencing reaction to called bases is feasible in a single channel.10 A set of compatible and automatable technologies were identified. A fused-silica capillary served as the microreactor of cycle-sequencing reaction inside a hot-air thermal cycler. A minibore chromatographic column based on size exclusion was used to purify the reaction products. A cross junction acted as a multifunctional device for denaturation, preconcentration, and injection at a high temperature. Capillary gel electrophoresis coupled with laser-induced fluorescence was utilized to read the DNA sequence. One of major obstacles toward multiplexing this approach is the inclusion of four bulky rotary valves. Most of the traditional mechanical valves, whether they are linear valves (gate, globe, diaphragm, pinch) or rotary valves (ball, plug, butterfly, shaft), are not suitable for constructing a highly parallel system. Yet, electroosmotic flow control is simply not compatible with the use of a purification column, in addition to unreliability under the changing buffer conditions necessitated by complex manipulations. Alternatively, a novel flow control principle first demonstrated by Bevan and Mutton11 has the potential for implementing fluid control without contamination in a multiplexed manifold. The advantages of this freeze/thaw valve (FTV) are noninvasive operation, zero dead volume, rapid response time, low cost, high-pressure tolerance, elimination of mechanical motion, and good electrical isolation. In this report, we constructed a set of automatic multiplexed freeze/thaw valves (MFTV) and demonstrated the feasibility of an eight-capillary on-line system performing microfluidics from DNA templates to called bases. Direct on-line injection to capillary array electrophoresis is made. The whole system is computer controlled with improved operation and regeneration protocols. The design allows for future expansion to a 96-capillary system. EXPERIMENTAL SECTION Reagents and Buffers. The 1× TBE buffer solution was prepared by dissolving a premix (Amerosco, Solon, OH) of 89 mM tris(hydroxymethyl)aminomethane (THAM), 89 mM boric (8) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (9) (a) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (b) Swerdlow, H.; Jones, B. J.; Wittwer, C. T. Anal. Chem. 1997, 69, 848-855. (c) Zhang, N.; Yeung, E. S. J. Chromatogr., B, in press. (10) Tan, H.; Yeung, E. S. Anal. Chem. 1997, 69, 664-674. (11) (a) Beven, C. D.; Mutton, I. M. J. Chromatogr., A 1995, 697, 541-548. (b) Beven, C. D.; Mutton, I. M. Anal. Chem. 1995, 67, 1470-1473.

Figure 1. Schematic of an assembly of multiplexed freeze/thaw valves and their arrangement in the experiment. The liquid nitrogen flow is controlled by cryogenic solenoid valves A and B. Valves U1 and U2 act simultaneously. When the temperature sensed by the thermocouple reaches 20 °C, the valves are considered open. When the temperature reaches -20 °C, they are considered off.

acid, and 2 mM ethylenediaminetetraacetic acid (EDTA) with 3.5 M urea in deionized water (pH ∼8.3). The 1× TE buffer solution was prepared by dissolving 10 mM THAM and 2 mM EDTA in deionized water, adjusted to pH ∼9.0 by 1.0 M HCl. Bovine serum albumin (BSA) and deionized formamide were from Sigma Chemical (St. Louis, MO). The fresh 5 mg/mL BSA solution was made daily by adding 100 mg of BSA to 20 mL of deionized water, stirring in an ultrasonic bath for 5 min, and degassing in a vacuum chamber. Methanol, anhydrous sodium hydroxide, and fuming hydrochloric acid were obtained from Fisher (Fairlawn, NJ). Urea was from ICN Biomedicals (Aurora, OH). Poly(ethylene oxide) (PEO) was obtained from Aldrich Chemical (Milwaukee, WI). The sieving matrix was made by dissolving 1.5% 8 000 000 MW PEO and 1.4% 600 000 MW PEO in 1× TBE buffer. The two kinds of PEO powders were first mixed well and slowly pulled into 1× TBE buffer while the mixture was being stirred violently. The violent stirring motion continued for 2 h and was then replaced by a slow stirring motion until all bubbles vanished and a uniform and clear gel was formed (∼8 h). 2% 1 000 000 MW poly(vinylpyrrolidone) (PVP) from Polyscience (Warrington, PA) in 1× TBE buffer was used to coat the separation capillaries between runs. Multiplexed Freeze/Thaw Valves. The three MFTV built into the fluidic network are shown in Figure 1. The MFTV was assembled on a 1/4-in.-o.d. stainless steel tube on which a small slot (or a set of eight small holes with 0.03-in. diameter) was opened on opposite sides of the center to allow passage of eight pieces of hypodermic tubes (0.028-in.-o.d. and 0.016-in.-i.d. stainless steel tubing from Small Parts, Inc., Miami Lakes, FL) packed in parallel. The hypodermic tubing array was then welded onto the 1/ -in. tubing. After a small K-type thermocouple (Omega, 4 Stamford, CT) was taped in the center of this array (without direct contact to the array), a heating tape (Omega) with 10 W/in.2 power density was wrapped around the 1/4-in. tubing and hypodermic tube array. A heat insulator was placed outside this assembly to prevent the loss of energy. A 250-µm-i.d. and 360-µm-o.d. fusedsilica capillary passed through each of the hypodermic tubes. To turn off the MFTV, the temperature was brought down to -20 °C by opening cryogenic solenoid valve A or B (Valcor Scientific, Springfield, NJ) and allowing liquid nitrogen to flow into the 1/4Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

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Figure 2. Schematic of the complete multiplexed and integrated instrumental design with eight capillaries. Stars at I, U1, and U2 represent the multiplexed freeze/thaw valves. The T-assembly is made up of eight pieces of commercial junctions stacked together. These connect to the manifold M1, the SEC purification columns, and the reaction loops. The cross assembly is made up of eight pieces of standard crosses packed together with built-in heaters. V8 is an eight-position motorized titanium valve with a center port. S1 is a two-position motorized PEEK valve. V6 is a six-position motorized PEEK valve.

in. tubing. A plug of solution inside each fused-silica capillary was thus frozen. To turn on the valves, the temperature was increased to 20 °C through the heating tape so that the solution plug was thawed. Note that there are eight simultaneous phase transitions in each MFTV whenever it changes its status. The signal from the thermocouple went through a thermocouple amplifier (OMNI-AMP-IV-13-115, Omega) and was converted into temperature after calibration. The temperature information, cryogenic valve, and heating tape form an automatic loop controlled with Labview. U1 and U2 in Figure 1 are synchronized, such that the temperature monitored at U2 was used for feedback and the two heaters were controlled by the same relay. So, the assemblies U1 and U2 can be considered as one 16-fold multiplexed on/off valve U, while valves I and U are completely independent. Instrumentation. Figure 2 shows the complete experimental setup for the multiplexed on-line sample preparation and purification microfluidic system coupled into capillary array electrophoresis for DNA sequencing. Microreactor Array. There are eight pieces of 77-cm-long, 250µm-i.d., and 360-µm-o.d. fused-silica capillary passing through the MFTV U1 and U2 and connecting to a T-assembly. Each capillary made a loop inside the hot-air thermocycler (Idaho Technology, Idaho Falls, ID) through a small hole drilled on the side of this machine. The T-assembly is a set of conventional HPLC Ts (Valco 4046 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

Instruments, Houston, TX) held together with a simple holder. The distance from the T-assembly to U2 is 8 cm (∼4 µL for each capillary), and the distance from the inlet of each capillary to U1 is 15 cm (∼7.5 µL). Also, the distances between each MFTV U and the hole on the thermocycler are ∼7 cm (∼7 µL). So, there is ∼40 cm (∼20 µL) of each capillary sitting inside the thermocycler. Lengths (15 cm) of 0.02-in.-i.d., 1/16-in.-o.d. Teflon tubing (∼30 µL) in each channel were used to connect the T-assembly with the manifold M1 (Valco Instruments). This arrangement is very important to guarantee quantitative injection to the purification columns later on. At the inlet end of this array, a holder was utilized to separate the capillaries to form a line and to align the tip of each capillary with the center of a row of a 96-well microtiter plate. Reaction mixtures can thus be directly loaded into the microreactor array from the microtiter plate. DNA polymerase (Thermo Sequenase, Amersham Life Science, Cleveland, OH) and rhodamine dideoxy dye-terminator kit (Applied Biosystems, Foster City, CA) were used to preformed dye-terminator cycle-sequencing reactions in the capillary microreactor array. A typical 20-µL reaction mixture in each channel is composed of 2 µL of 2.0 mg/mL BSA, 4 µL of ddNTP mixture (0.22 µM ddATP, 2.0 µM ddCTP, 0.1 µM ddGTP, 2.75 µM ddTTP), 2 µL of dNTP mixture (3.0 mM dATP, 3.0 mM dCTP, 3.0 mM TTP), 3.6 µL of 5.0 mM dITP (Sigma Chemicals), 1.6 µL of reaction buffer (260 mM Tris-HCl and 65 mM MgCl2 at pH ∼9.5),

2 µL of Thermo Sequenase dilution buffer (10 mM Tris-HCl, pH 8.0, 1 mM 2-mercaptoethanol, 0.5% Tween-20, 0.5% Nonidet P-40), 0.5 µL of Thermo Sequenase storage buffer (32 units/µL Thermo Sequenase, 20 mM Tris-HCl, pH 8.5, 50% glycerol, 0.1 mM dithiothreitol, 100 mM KCl), 1 µL of 5.0 µM ) 40M13 universal primer, 1.7 µL of 0.2 µg/µL M13mp18 ss-DNA in 1× TE buffer (pH ∼7.5, Amersham), and 1.6 µL of deionized water. The temperature protocol here is similar to the one we described previously. Reaction mixtures were heated to 96 °C and held for 2 min; 30 thermal cycles were performed with denaturation at 96 °C for 10 s, annealing at 45 °C for 5 s, and extension at 60 °C for 4 min; and then the sample was ramped to 95 °C and held for 3 min. Pumping and Regenerating System. A 15-cm-long, 0.76-mmi.d., and 1.6-mm-o.d. Teflon tube connected the manifold M1 with an eight-position column selection titanium valve V8 (Valco Instruments, Inc.). The ports of V8 were connected to a waste line, a manual syringe, and regeneration reagent reservoirs including deionized water, methanol, 1× TE buffer, and 0.2 M NaOH solution. These reagents are used to regenerate both the microreactor array and the purification columns. The center port of the eight-position valve V8 was coupled to a two-position sample injection valve S1 (Rheodyne, Cotati, CA) through a 10-cm-long, 0.03-in.-i.d. and 1/16-in.-o.d. Teflon tube. One position of S1 was connected to a micropump (Ultra-plus MicroLC System, MicroTech Scientific, Sunnyvale, CA) and the other to a port of a sixposition selection valve V6. The main line of V6 was connected to a syringe pump drive module (Kloehm Co., Inc., Las Vegas, NV) with a 1-mL syringe. The other ports of V6 were connected to the reservoirs of running buffer (1× TBE), deionized water, BSA solution, waste bottle, and the center port of the manifold M2. All valves and pumps used here can be activated by an electronic signal from the computer, as described in the Automation and Control section. Purification System. The procedure for packing the purification columns with size-exclusion media (Sephadex G-25-50, Supelco, Bellefonte, PA) was described previously10 except that the column frit was changed into an in-line filter from Upchurch Scientific (Oak Harbor, WA). Care needs to be taken to obtain eight uniformly packed columns with similar capacity. The inlets of these columns were connected to the T-assembly through 20-cmlong, 150-µm-i.d., and 360-µm-o.d. fused-silica capillaries while the outlets were coupled with 100-µm-i.d. and 360-µm-o.d. fused-silica capillaries to a cross assembly for injection. The lengths of these 100-µm-i.d. capillaries varied between 50 and 60 cm for different channels so that the small difference in flow rates among channels can be compensated in this way. Most of the length of these 30cm-long SEC columns (except their frits) were immersed in a hot water bath (HB, Grant Instruments, Barrington Cambridge) which is preset at 60 °C. A 1-mW 543.6-nm HeNe laser (Melles Griot, Irvine, CA) was used for monitoring the elution from one of the purification columns at point A. An uncoated plano-convex lens (Edmund Scientific, Barrington, NJ) with 12-mm focal length was used to focus the laser to the capillary window. A 10× microscope objective (Edmund) was used to collect the fluorescence perpendicular to the excitation laser. A RG610 cutoff filter was employed to block the scattered light. A photomultiplier tube (R928,

Hamamatsu Corp., Bridgewater, NJ) operating at 1000 V was used to generate an electrical signal. The signal was digitized at one of the analogue input channels of a multifunctional data acquisition board (AT-MIO-16DE-10, National Instruments, Austin, TX). Multiplexed Interface to Capillary Array Electrophoresis. The cross assembly was composed of eight PEEK crosses (Upchurch Scientific), a heating tape (Omega), a themocouple (Omega), aluminum rods (eight pieces with 1-in. diameter and 1/4 in. long and one piece with same diameter but 1.5 in. long). Each cross was sandwiched between two pieces of short aluminum rods, and the whole multilayer sandwich was mounted on the long aluminum rod and a plastic holder. The heating tape simultaneously wrapped around all the aluminum rods so that they became nine similar small heaters. The thermocouple was implanted in the center of this assembly. Meanwhile, an electrical fan can blow air into the whole assembly for cooling. The four limbs of each cross were respectively connected into the purification column, manifold M2, the separation capillary, and manifold M3. The connection made to M3 was through a short piece of 2-in.-long, 0.03-in.-i.d., and 1/16-in.-o.d. stainless steel tube and a long piece of 12-cm-long Teflon tube with the same inner diameter. All the stainless steel tubes used here were welded to a piece of copper wire, which acts as the ground electrode while electrophoresis proceeds. The connections between the manifold M2 and M3 must be made as symmetrical as possible in order to get uniform flow rates at each channel during the run. Teflon tubing was also used to bring the waste stream from M3 to a waste bottle outside. The whole cross assembly was horizontally placed with the manifold M2 at the bottom and the manifold M3 at the top. The cross assembly provides four major functions in the whole instrument. First, it interfaces the purification columns with the separation capillaries to allow a heart-cut injection to proceed. Second, it can raise the temperature to facilitate DNA sample denaturation while injecting (so-called hot injection). Third, it supplies both separate 1× TBE buffer flows to each channel during separation and collects waste from the purification columns and the transverse flows from the syringe pump. Finally, it also maintains electrical contact for each channel for electrokinetic injection and electrophoretic separation of DNA fragments. DNA Separation and Detection. Eight separation capillaries with 75-µm i.d. and 360-µm o.d. were packed side by side at the window region. Windows were constructed by burning the polyimide coatings of each capillary with a microtorch, removing burrs at both sides of the window with a blade, and clamping those between two flat surfaces of a plastic holder. The plastic holder was then mounted on a translational stage after the capillary array was adjusted and tilted to be parallel to the optical table. The detection ends of the capillaries were separately immersed into 1× TBE buffer while the injection ends were connected to the cross assembly. The geometry for multiplexed excitation and detection in this work is similar to the previous works by Kambara’s16 and Yeung’s17 groups although minor modifications were made to further (12) (a) Guttman, A.; Schwartz, H. E. Anal. Chem. 1995, 67, 2279-2283. (b) Chien, R.-L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (13) Mizusawa, S.; Nishimura, S.; Seela, F. Nucleic Acids Res. 1986, 14, 13191324. (14) Barr, P. J.; Laybourn, P.; Najarian, R. C.; Seela, F.; Thayer, R. M.; Tolan, D. R. BioTechniques 1986, 4, 428-432.

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improve S/N ratios. Briefly, 15 mW of 514.5-nm light from an air-cooled single-line argon ion laser (Uniphase, Palo Alto, CA), after being separated from its plasma emission by a 60° prism (Edmund Scientific), was focused on the first capillary of the array through a 40-mm focal length plano-convex lens (Edmund Scientific). The laser beam not only was coplanar with the capillary array but also had a 20° incident angle to the array at point B. The multiple laser focusing described by Kambara16 still holds in this case and can be judged by looking at the image spot of the laser after the array. The off-axis arrangement avoids collecting the dominant scattering ring into the CCD camera to lower the background. Fluorescence from DNA bands in each capillary was monitored simultaneously with a cooled CCD camera (Photometrics, Tucson, AZ) from a direction perpendicular to the capillary array plane. The CCD camera had a lens (Canon, Japan, 70-mm diameter and 24-mm focal length) attached, two holographic notch-plus filters at 514.5 nm (Kaiser Optical System, Ann Arbor, MI) behind the lens, and an image-splitting filter set before the lens. The imagesplitting filter set was made of a 630-nm long-pass filter (Edmund Scientific) and a quartz plate to compensate for the optical path length with a tilted angle. Two images were formed corresponding to each capillary with an image size of 7 × 3 pixels. The image from the 630-nm filter is called the red channel while the one from quartz plate is the blue channel. This two-color detection scheme has been reported previously.10 The exposure time of the camera is set at 300 ms, so the frame rate is ∼3 Hz. Each frame containing two subarrays of pixels for both blue and red channels was transferred into a host computer (Pentium 133 MHz) during the exposure. Frames were accumulated into a raw data file by software written in C++. After the run, the raw data file was then extracted into an ASCII file at the pixel corresponding to the center of each capillary. A base-calling program developed in Labview based on the histogram of four-label two-color ratios was then used to identify the sequence. Bare fused-silica capillaries (75-µm i.d., 365-µm o.d.) (Polymicro Technologies, Phoenix, AZ), typically 80 cm long (60 cm effective length) were used as separation capillaries. The separation capillaries were flushed and coated with 2% 1 000 000 MW PVP in 1× TBE buffer before filling with the sieving matrix. PEO was transferred and forced into the capillaries through a piece of 0.015in.-i.d. Teflon tubing attached to a 100-µL syringe from the detection end of each capillary. After running, the capillaries were simply flushed with 0.1 M HCl solution and deionized water or pure methanol and then coated with 2% PVP solution again. A high-voltage power supply (Spellman, Plainview, NY) was used to drive the electrophoresis from the anode at the detection end. Typically, 150 V/cm was used for both separation and injection of DNA Sanger fragments. Automation and Control. A Dell GXMT 5100 computer (Dell Computer Corp., Austin, TX) equipped with a multifunctional data acquisition board (AT-MIO-16DE-10, National Instruments) and a two-port serial interface board (AT-232/2, National Instruments) (15) (a) Van der Moolen, J. M.; Poppe, H.; Smit, H. C. Anal. Chem. 1997, 69, 4220-4225. (b) Figeys, D.; Ning, Y.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (16) Anazawa, T.; Takahashi, S.; Kambara, H. Anal. Chem. 1996, 68, 26992704. (17) Lu, X.; Yeung, E. S. Appl. Spectrosc. 1995, 49, 605-609.

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Figure 3. Computer control protocol for multiplexed system in Figure 2.

were used as control hardware while Labview (National Instruments) was the programming platform for control software. The schematic is shown in Figure 3. Two serial ports originated from the Dell computer and two additional ports from the AT-232/2 board provide four communication channels through RS-232 protocol. They were employed to control the syringe pump and its selection valve V6, the micropump controller, selection valve V8, and 1/16 DIN temperature controller. The temperature of the cross assembly was governed by the 1/16 DIN temperature controller through the heating tape, cooling fan, and thermocouple. The analog input signals include the PMT for monitoring chromatographic elution, two thermocouples at the MFTV, and the total current of electrophoresis. Lines of digital I/O were connected to relays (ER/16, National Instruments) for controlling the cryogenic valves, heating tapes for MFTV, heater for the SEC purification column, and contact closure control of the sample injection valve S1. A parallel cable was used to transfer the ASCII data files from the CCD host computer to the Dell computer for base calling. This instrumental prototype is completely controlled by the computer except for gel loading and operation of the hot-air thermocycler. Current efforts in commercializing the multiplexed capillary DNA sequencer will provide automation in the gel loading and column regeneration processes. A new version of the hotair thermocycler is available to allow full computer control. Operation and Regeneration Protocols. The instrumental operation can be divided into four steps, which are loading of cyclesequencing reaction mixture into the microreactor array, loading of these reaction products into the SEC purification columns, heart-cut injection into multiplexed capillary array, and size-based separation and detection of the sequencing ladder. The regeneration protocols for reaction capillaries, purification columns, separation capillaries, injection assembly, and syringe pump were tested step by step throughout the project. Figure 4 shows the key steps to load samples separately into the reaction loops and the purification columns before and after the Sanger reactions. With valve I closed and valve U opened, 22 µL was aspirated into each reaction capillary from the microtiter plate where a row of premixed templates and reagents was placed. Then, another plug of 8.5 µL of deionized water was loaded into each capillary. This placed the reaction plug in each capillary

The microreactor array, the cross assembly, and the Tassembly were simply regenerated by flushing with 0.2 M NaOH solution, 1× TE buffer, methanol, and deionized water sequentially. The purification columns can be recovered by NaOH solution and 1× TE buffer as described previously. The syringe pump was always rinsed by two full strokes of water and the loading reagent whenever it exchanged reagents. All the regeneration protocols carried here were automated by controlling the syringe pump except that the regeneration of the separation capillary array was still done manually with a 100-µL syringe.

Figure 4. On-line operation protocol from reaction capillary to purification column. g, Open star means MFTV is on. f, Filled star means MFTV is off. Wave filling represents the buffer or water. Dot filling denotes the reaction mixture. (A) Simultaneous reaction and regeneration of SEC column. During cycle sequencing reaction, the 1× TE buffer flushes the purification column with valve U off. (B) Loading. When reaction is complete, the syringe pump aspirates the reaction products above the T-assembly with valve I off. (C) Micropump drives the Sanger products into the purification columns at 20 µL/min per channel with U closed.

right inside the thermal cycler. After opening valve I and closing valve U, the cycle-sequencing reactions were started (Figure 4A). During reaction, 1× TE buffer continuously flushed the purification columns at a flow rate of 20 µL/min per channel from the micropump. After reaction, valve I was closed and valve U was opened, so that 22.5 µL/channel was pulled into the Teflon tubing region above the T-assembly (Figure 4B). After switching off valve U and switching on valve I, reversing the flow direction drove these plugs into the purification columns (Figure 4C). Purification was performed at a flow rate of 20 µL/min per channel with 1× TE buffer at 60 °C as controlled by the hot water bath. Once the purification step started (by selecting the micropump at valve S1), there were two operations necessary to prepare the cross-assembly junction for injection. The temperature at the cross assembly was set at 70 °C while deionized water flowed over the injection junction at 200 µL/min from the syringe pump. The continuous transverse flow of water at the injection end of the separation capillaries causes dialysis at the interface of the sieving matrix to create a low ionic strength zone. This promotes stacking injection for preconcentration of the analytes. When the Sanger fragments reached the LIF detector at point A, as recognized by the onset of a peak, electrokinetic injection to the separation capillaries was carried out at 150 V/cm for 2 min. When injection was completed at about one-third of the peak height on its falling edge, valve I was turned off and valve S1 was also switched to the syringe pump position. One milliliter of 1× TBE buffer was used to clean out the deionized water and reionize the injection zone at a very low flow rate to avoid losing the injected fragments. The 1× TBE transverse flow was then programmed to 500 µL/min per channel during electrophoretic separation at 150 V/cm. More importantly, the temperature at the injection cross assembly needs to be lowered to room temperature as quickly as possible after restoring the ionic strength at the cross junction. The MFTV valve I can be turned on and regeneration of the purification columns and reaction capillaries can proceed after the electrophoresis current has stabilized (∼40 min).

RESULTS AND DISCUSSION In the course of developing a highly multiplexed and on-line integrated system for DNA analysis, we found that the major barrier in our previous study10 is the use of the rotary valves to control flow. The strategy to solve this problem is to design a distribution network that reduces the number of parallel valves and that replaces the rotary valves with on/off valves. Then, multiplexing the on/off valves can be accomplished according to the freeze/thaw principle.11 We have successfully used a singlechannel system to test the key operational protocols shown in Figure 4. The sequence for pGEM template can be called up to 550 bp with 98% accuracy. However, when scaled up to an eightchannel system, the many technical challenges are magnified. Multiplexed Freeze/Thaw Valves. To test the performance of MFTV valves, electrical currents for capillary electrophoresis were used as indicators, as reported by Bevan and Mutton.11 When a valve is closed, an ice plug forms inside the capillary, halting electroosmosis and electrophoresis and also stopping the current flow. Four pieces of 80-cm-long, 250-µm-i.d., and 360-µm-o.d. fused-silica capillaries filled with 1× TBE buffer under 10 kV were placed at the edge of valve U1, the center of valve U1, the edge of valve U2, and the edge of valve I, respectively. The current from each capillary was monitored through a 15-kΩ resistor. We found that the response time on closing is about 20-25 s while the response time on opening is about 35-45 s. As expected, the response time for the initial closing of MFTV is always the longest because the inlet tubes need to be cooled. The response time differences among the MFTV were relatively small, less than 1 s within valve U1 and ∼4 s between valve U1 and valve U2. The response time for MFTV highly depends on the design and thermal conductivity of the materials used. Our cross-flow design not only prevents the fragile capillaries from being broken once frozen but also sends the waste nitrogen gas out of the instrument. The response times achieved with this design suffice for the purpose of control of DNA sequencing although there is room for improving the speed with better engineering. These results show that the minimum time for loading the reaction products from the microreactor array into the purification columns is 3 min. This limitation should not cause any degradation in the performance of the purification columns. Even if it does, it can be easily corrected by injecting for a longer time. The principle of multiplexed on/off switching demonstrated here with the phase transition of water is readily scalable up to 96 channels without any modifications or negative effects on their performance. The physical size of the current MFTV design is suitable for even larger arrays. For 96 capillaries, one only has to use a rectangular tubing with 3.6-cm-wide by 370-µm-high inner Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

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dimensions to replace the hypodermic tube array in Figure 1. With better thermal contact and higher liquid nitrogen flow, 1000 valves operated in parallel should also be manageable in a relatively small assembly. Flow Uniformity. Achieving uniform flow rates in each channel is very critical to success of this multiplexed operation. The accuracy of sample loading, the timing of “heart-cut” injection at the cross junction, and the similarity of transverse flow at the interface between the purification columns to the separation capillaries all depend on how uniform the flow rates are among the channels. According to Pouiselle’s law, the flow rate in a tube is mainly dependent on the pressure drop, length, and average inner diameter, temperature, and viscosity of the fluids inside. Here, the capillary inner diameter, pressure drop, temperature, and viscosity among the channels are essentially identical. Therefore, the capillary length is the most important parameter toward achieving a uniform and controllable flow rate. In the microreactor array, the variation in flow rates can be experimentally determined by the difference in aspirated lengths of liquid plugs viewed outside the capillaries. We found there is ∼2% relative standard deviation among the channels. So, 20 µL of reaction mixture occupies 40 ( 2.4 cm of capillary, the required distance between the two U valves. A distance of 7 cm was thus introduced between the MFTV valves U. For the same reason, 30 µL of Teflon tubing between the manifold M1 and the T-assembly was used to guarantee independent sample manipulation in the eight channels. Although 22.5 µL of liquid plug was loaded into this Teflon tube region, only 15 µL of sequencing products was injected into the purification column. The other 7 µL of deionized water was also loaded into the purification column, but there are no observable negative effects in terms of purification and regeneration. In the purification columns, the main source of back pressure is the restricted channel spacing between the packed particles, not the inner diameter. If the back pressures of the individual channels are not identical, flow will favor the channels with the least flow resistance. However, the Sanger products have large sizes and should not be retained in size exclusion chromatography. They are eluded in the void volume. The denser the column packing, the smaller its void volume. This may balance out the higher back pressure in a given column and thus the lower flow rate. Moreover, the arrival time can also be adjusted by manipulating the capillary length after the purification column. Figure 5 shows the uniformity of elution monitored at point A. It is clear that a synchronous heart-cut injection can be performed with careful engineering and relatively uniform column packing. Once the system has been calibrated, only one of the purification columns needs to be monitored. In Figure 6, the upper panel shows the chromatogram from one purification column and the lower panel shows the total current profile while heart-cut injection is performed. The regular spikes are due to pulsations from the micropump. The peak width is ∼3 min at 20 µL/min per channel flow rate. We can estimate the dilution factor to be ∼0.25. A 2-min electrokinetic injection at the cross junction will sample 96% of the eluting plug. The DNA fragments are focused at the entrance, and the CE resolution is preserved. This phenomenon has been reported and used to preconcentrate samples previously.12 In principle, asynchronous 4050 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

Figure 5. Simultaneous chromatograms for purification of cycle sequencing products. The total flow rate before the manifold M1 is 160 µL/min. Fluorescence data were acquired via excitation by a 5-mW 514.5-nm Ar+ laser at 300-ms exposure time with a CID detector after passing through a 610-nm long-pass filter. Capillaries (90-cm-long and 100-µm-i.d.) were used to connect to the purification columns.

Figure 6. Illustration of heart-cut injection operation showing both the signal from laser-induced fluorescence (a) and the total injection current response (b). The regular spikes are associated with pulsations in the micropump.

injection can be carried out by separately monitoring the Sanger fragments in each channel but was found to be unnecessary. This simplifies expansion of the number of capillaries to 96 or even 1000. We found that the back pressure developed at the purification column is less than 50 psi/channel. As far as the pressure is concerned, scale-up of the operation does not require higher pressure simply due to the proportional increase of the cross-sectional area. However, the pumps will have to have larger bores to handle the increased (total) liquid volume. Injection Protocols. The injection temperature must be optimized to denature the Sanger fragment at the cross junction without creating any air bubbles in the capillary. With dITP cyclesequencing chemistry,13 70 °C is perfect for both denaturation and injection. With 7-deaze-dGTP chemistry,14 90 °C should be used for denaturation such that bubbles can form during the CE run to block the capillary, although it allows for increased read length compared to dITP. As for multiplexing injection, even temperature across the cross assembly is required. With the current design, it is difficult to scale-up further due to the physical size of commercial crosses. A microfabricated device directly coupling with the capillaries15 is a potential solution for future versions of this instrumentation. We found that it is not necessary here to

Figure 7. Electropherograms showing simultaneous sequencing of eight M13mp18 DNA templates with the on-line integrated and multiplexed system starting from templates. Conditions: Reaction plug, 40 cm out of 77 cm of 250-µm-i.d. and 360-µm-o.d. fused-silica capillaries; flow rate, 20 µL/min per channel; purification, Sephadex G-25-50 columns set at 60 °C; injection, heart-cut injection at 70 °C, with ∼2 mL of water preconditioning; separation, 60-cm effective length of bare capillary filled with 1.5% high-molecular-weight PEO and 1.4% low-molecular-weight mixture, 150 V/cm; detection, CCD camera; excitation, 15 mW 514.5-nm Ar ion laser with exposure time of 300 ms. Capillaries in the array are labeled from 1 to 8 according to proximity to the excitation laser.

Figure 8. Reproducibility of S/N and resolution in the array.

coat the cross junctions with BSA solution, in contrast to our previous study.10 This may be a result of differences in the

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Figure 9. Sequencing of M13mp18 DNA from one of the capillaries in Figure 7. The raw data from the blue and red channels are plotted. The miscalls are also corrected under the corresponding bases.

than electroosmotic flow. This is necessitated by the presence of a purification column. Also, the various solutions that have to be employed to purify, rinse, run electrophoresis, and recondition the components will cause the nature of the surfaces to change constantly over the course of the operation. Electroosmotic control of the flow is simply not possible. Performance. The electropherograms (raw data) at the blue channel from a completely multiplexed and on-line run starting from M13mp18 templates are shown in Figure 7. Each electropherogram was normalized to the span of its panel in the Gram/ 386 software. The absolute fluorescence intensities at the same 4052 Analytical Chemistry, Vol. 70, No. 19, October 1, 1998

base number decreased gradually with the capillary number. This is an inherent feature of the side-entrance excitation geometry reported by Kambara’s16 and Yeung’s17 groups. We found much larger variations of intensities among capillaries than that reported by Kambara.16 This fluctuation is caused by heterogeneity in the efficiencies of individual sequencing reactions, efficiencies of the stacking injection, and temporal variations in the heart-cut injection. All electropherograms, however, have very high S/N ratios, as can be judged from the baselines before the primer peaks in Figure 7. The S/N of the blue channel for capillary 8 is the lowest, but is still 39 at 453 bases, which is clearly enough to call bases.

The migration time and resolution of DNA fragments in each capillary were not completely uniform among the capillaries even for the best three shown in Figure 8. This is probably attributable to inhomogeneities in the gel matrix, variations in capillary length, and reproducibility of the (separation) capillary regeneration and coating procedures. Naturally, in DNA sequencing, each channel is an independent experiment and quantitative reproducibility is not required. For all eight capillaries in Figure 7, sequences can be called based on the raw data up to 400 bases with an accuracy of 98%. The best capillary produced this same level of accuracy out to 460 bases (at 120 min). Figure 9 shows the DNA sequence from this capillary in the eight-capillary array. The y-axis is the relative fluorescence intensity normalized to the maximum peak in the electropherogram. The x-axis is the migration time in terms of data points, starting at 37.2 min for 0 and collected at 2.8 points/ s. The intensities of the G’s are low due to the use of a 630-nm cutoff filter, poor enzyme incorporation rate, and the lower ddGTP concentrations. The gaps between bases were corrected by software to account for these weak G’s. The last (compression) peaks come out at ∼150 min with 60-cm effective capillary length and 150 V/cm. Actually, only about half the capillary length is necessary for separation. With better software and further optimization of the PEO sieving matrix and separation conditions, it should be possible to sequence 500 bases within 1 h in the current setup. The ability to recondition the multiplexed system is a major issue. We were able to use the same microreactor array and the same set of purification columns over a period of 4 months for more than 20 runs. We have not yet observed any significant degradation in performance. Although the reaction efficiency may vary from run to run and from capillary to capillary, it is not critical for DNA sequencing as long as it provides sufficient sequencing products to maintain a minimum S/N. Multiplexing did not add any extra requirements to the regeneration protocols that were demonstrated before.10 On the other hand, multiplexing did add a small positive pressure to the separation capillaries due to the presence of a transverse buffer flow during electrophoresis. This is not a problem for integration with a single channel because the flow-gate interface18 has been successfully employed even in capillary zone electrophoresis. The difference in multiplexing is the need to produce uniform transverse flow rates among the capillaries, which requires a finite flow resistance at the waste outlet of the cross assembly. This (18) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (19) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388. (20) Madabhushi, R. S. Electrophoresis 1998, 19, 224-230. (21) Swerdlow, H.; Dew-Jager, K.; Gesteland, R. F. BioTechniques 1993, 512519. (22) Salas-Solano, O.; Carrilho, E.; Kotler, L.; Sosic, Z.; Miller, A.; Karger, B. L. Eleventh International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques, February 1-5, 1998, Orlando, FL, Abstract P547.

means that a relatively nonviscous sieving matrix may be disturbed by the flow to degrade the resolution. So, the system has the potential limitation of being incompatible with newly developed sieving matrixes such as PVP19 (4.5% with 27 cP) and poly(dimethylacrylamide)20 (PDMA, 6% with 75 cP) because of their extremely low viscosities. The turnaround time for this demonstration is ∼5.5 h, including 2.5 h for separation, 2.5 h for reaction, and 0.5 h for purification and sample loading. The regeneration of the reaction capillaries can be done during separation and vice versa. With a slight modification of the protocol described here, one should be able to carry out the Sanger reaction and electrophoretic separation at the same time in the two subunits of the system. Furthermore, the reaction time in the air thermal cycler can be as short as 25 min.21 Reducing the separation time to 1 h with 1000 bases sequenced has also been reported recently.22 Producing 1.2 million bases of sequencing data per day per instrument should therefore be achievable if the system is scaled up to 96 channels. CONCLUSIONS A completely integrated and multiplexed on-line DNA sequencer, which allows one to process multiple DNA samples from template to called bases, has been demonstrated. A set of compatible technologies was identified for coupling the Sanger reaction to the detection system directly. The multiplexed freeze/ thaw valves provide a relatively flexible way to control liquid flow simultaneously in many capillaries. Optimization of the injection and separation conditions led to longer readable sequencing data due to better S/N and better resolution in DNA separation compared with that in our previous study. A total of 460 bases can be read out with an accuracy of 98%. To our knowledge, this is the first demonstration of multiplexed operation from DNA sample preparation to called bases. Although this is only an eightcapillary system, it is readily scalable to a 96-capillary on-line system. Future developments should allow even more accurate handling of the fluidics, miniaturization of the reaction volume to reduce the cost of reagents, and adaptation to other genotyping and PCR analysis. Even standard chemical manipulations prior to CE can be handled in this manner to achieve high-throughput analysis in the routine chemical laboratory. ACKNOWLEDGMENT The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract W-7405-Eng-82. This work was supported by the Director of Energy Research, Office of Health and Environmental Research, and by the National Institutes of Health through Grant HG-01385. Received for review April 13, 1998. Accepted July 28, 1998. AC980406I

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