Integrated On-Line System for DNA Sequencing by Capillary

An integrated on-line prototype for coupling a micro- reactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled ter...
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Anal. Chem. 1997, 69, 664-674

Integrated On-Line System for DNA Sequencing by Capillary Electrophoresis: From Template to Called Bases Hongdong Tan and Edward S. Yeung*

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

An integrated on-line prototype for coupling a microreactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled terminator cyclesequencing reaction is performed in a fused-silica capillary. Subsequently, the sequencing ladder is directly injected into a size-exclusion chromatographic column operated at ∼95 °C for purification. On-line injection to a capillary for electrophoresis is accomplished at a junction set at ∼70 °C. High temperature at the purification column and injection junction prevents the renaturation of DNA fragments during on-line transfer without affecting the separation. The high solubility of DNA in and the relatively low ionic strength of 1× TE buffer permit both effective purification and electrokinetic injection of the DNA sample. The system is compatible with highly efficient separations by a replaceable poly(ethylene oxide) polymer solution in uncoated capillary tubes. Future automation and adaptation to a multiple-capillary array system should allow high-speed, high-throughput DNA sequencing from templates to called bases in one step. Highly multiplexed capillary electrophoresis (CE), especially when combined with replaceable linear polymer solutions, for DNA sequencing has been demonstrated by many research groups.1-6 The attractive features of such a system have been illustrated in terms of the speed, throughput, resolution, reliability, and sensitivity compared to current DNA sequencing instrumentation, particularly for sequencing the human genome. At present, highly multiplexed CE imposes a great demand on the throughput of sample preparation for DNA sequencing. Linear amplification sequencing (cycle sequencing), which is the Sanger dideoxy termination chemistry modified by the concept of polymerase chain reaction (PCR), has gained popularity due to the lower DNA template requirement, ease of automation, and effective denaturation of double-stranded DNA.7,8 The marriage of automated cycle sequencing with highly multiplexed capillary array electrophoresis (1) Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424-1431. (2) Swerdlow, H.; Gesteland, R. F. Nucleic Acids Res. 1989, 18, 1415-1419. (3) Cohen, A. S.; Najarian, D. R.; Paulus, A.; Guttman, J. A.; Karger, B. L. Proc. Nat. Acad. Sci. U.S.A. 1988, 85, 9660-9663. (4) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; DiCunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903. (5) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Grey, R.; Wu, S.; Harke, H. R.; Dovichi, N. J.; Fuller, C. Anal. Chem. 1991, 63, 2835-2841. (6) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967972. (7) Murray, V. Nucleic Acids Res. 1989, 17, 8889-8995. (8) Craxton, M. Methods (San Diego) 1991, 3, 20-26.

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has the potential to further increase the throughput and reduce the cost in large-scale DNA sequencing projects. Numerous endeavors have been made toward developing robotic workstations to perform the sequencing reaction, purification, preconcentration, denaturation, and sample loading in slab gel electrophoresis (SGE).9,10 Although robotization has shown advantages in repetitive operation with high precision, the adaptation to highly multiplexed capillary array separation and detection suffers from many incompatibilities in terms of the total reaction volume, purification by centrifugation, lyophilization, and sample injection after reconstitution and denaturation. The small sample requirement of CE, typically ∼5 nL, provides a great opportunity to reduce the amount of DNA template and reagents, leading to a substantial reduction of the cost per base pair. Miniaturization of cycle sequencing in a glass capillary has been demonstrated.11 PCR reaction in a capillary format has recently been achieved in only ∼1.5 µL of total volume.12 Moving the cycle-sequencing reaction into a capillary has the additional advantage of increasing the reaction speed due to the small heat capacity of a capillary vs a heating block or a water bath. A capillary reactor is also compatible with highly multiplexed electrophoresis in a parallel capillary array. A microchip providing integrated operation from Sanger reaction to sequencing separation is a promising approach, but such has not yet been demonstrated. Many DNA purification methods such as ultrafiltration, spincolumn gel filtration, acetate-ethanol precipitation and phenolchloroform extraction require a centrifuge to remove fluorescently or radioactively labeled primers or terminators in cycle-sequencing strategy. Interfacing the centrifuge into the robotic workstation complicates the whole sequencing protocol. A vacuum chamber under the microtiter plate has been used to replace the centrifuge in a robotic workstation.13 Solid-phase magnetic beads have also been utilized to bind the cycle-sequencing products.14 Almost all these modifications still depend on the complicated combination of multiple movements of a robotic arm and precise dispension (9) Wilson, R. K.; Yuan, A. S.; Clark, S. M.; Spence, C.; Arakelian, P.; Hood, L. E. BioTechniques 1988, 6, 776-777. (10) Zimmermann, J.; Voss, H.; Schwager, C.; Stegemann, J.; Angsorge, W. FEBS Lett. 1988, 233, 432-436. (11) (a) Swerdlow, H.; Dew-Jager, K.; Gesteland, R. F. BioTechniques 1993, 15, 512-519. (b) The same group also reported coupling of the reaction capillary to CE at the HPCE ’94 and FACSS ’94 meetings. (12) Meldrum, D. R.; Seubert, R. C.; Kraft, R. H.; Wiktor, P. J.; Friedman, N. Third International Conference on Automation in Mapping and DNA Sequencing, Berkeley, CA, 1995; p 37. (13) McCombie, W. R.; Heiner, C.; Kelly, J. M.; Fitzgerald, M. G.; Gocayne, J. D. DNA Sequence/DNA Sequencing Mapping 1993, 2, 289-296. (14) Tong, X.; Smith, L. M. Anal. Chem. 1992, 64, 2672-2677. S0003-2700(96)00892-X CCC: $14.00

© 1997 American Chemical Society

on the robotic platform. The inability to miniaturize the robotic workstation restricts further miniaturization and multiplexing of the sequencing reactor to couple to CE arrays. An alternative concept to purify the sequencing reaction products is on-line chromatographic separation. Size-exclusion chromatography (SEC)15 has the unique features of high recovery, desalting ability, favorable elution order for subsequent injection and cleanup, and suitability for pressure flow instead of centrifugation. The online coupling of SEC with CE should also allow multiplexed injection into a CE array. In this paper, we demonstrate an integrated on-line protocol for interfacing cycle-sequencing reactions to capillary electrophoresis. A fused-silica capillary is used as the microreactor. An on-line Sephadex column is utilized to purify the sequencing reaction products from the fluorescently labeled dideoxynucleotides (terminators). We also demonstrate the on-line denaturation and injection of the DNA sequencing ladder into CE. Factors affecting the many individual steps in the process are also examined. Compromises are made to allow complete integration of this system. EXPERIMENTAL SECTION Reagents, Buffers, and Separation Matrix. All chemicals for preparing buffer solutions were purchased from ICN Biochemicals (Irvine, CA). Bovine serum albumin (BSA)and deionized formamide were from Sigma Chemical (St. Louis, MO). Methanol, anhydrous sodium hydroxide, and fuming hydrochloric acid were obtained from Fisher (Fairlawn, NJ). Poly(ethylene oxide) (PEO) was received from Aldrich Chemical (Milwaukee, WI). The 1× TBE buffer solution was prepared by dissolving 89 mM tris(hydroxymethyl)aminomethane (THAM), 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA), and 3.5 M urea in deionized water (pH ∼8.3). The 1/100× TBE buffer solution was made by diluting 1× TBE buffer (without urea) by a factor of 100 with deionized and autoclaved water. The 1× TE buffer solution was prepared by dissolving 10 mM THAM and 1 mM EDTA in deionized water (pH ∼8.2 by adding NaOH). 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. The sieving matrix was an entangled polymer solution made by dissolving 1.5% of 8 000 000 MW PEO and 1.4% of 600 000 MW PEO in 1× TBE buffer. The bare fused-silica capillary, typically 60 cm long (45-cm effective length), was treated sequentially with pure methanol, deionized water, and 1 M hydrochloric acid before filling with PEO matrix. The preparation procedure for the PEO matrix and the regeneration of capillary columns were described previously.16 Sequencing Reaction Protocol. Sequencing reactions were performed inside a hot-air thermal cycler (Rapidcycler from Idaho Technology, Idaho Falls, ID). Either Thermo Sequenase from U.S. Biochemical (Cleveland, OH) or AmpliTaq FS polymerase from Applied Biosystems (ABI, Foster City, CA) was used in a dye-terminator cycle-sequencing protocol. Modifications were made to fit the protocol to a capillary format since the original protocol was developed for the ABI Model 9600 or 2400 instrument. (15) Hagel, L.; Janson, J.-C. J. Chromatogr. Libr. 1992, 51A, A267-A307. (16) Fung, E. N.; Yeung, E. S. Anal. Chem. 1995, 67, 1913-1919.

Bovine serum albumin was added to prevent surface denaturation of the enzymes on the surface of capillary tubes.17 A typical cycle-sequencing reaction mixture for Thermo Sequenase consisted of 1.25 µL of 0.2 µg/µL M13mp18 ss-DNA in 1× TE buffer (pH ∼7.5, Amersham), 3.0 µL of 1.78 pmol/µL universal primer (23-mer, 5′-GTTTTCCCAGTCACGACGTTGTA-3′), 1.6 µL of reaction buffer containing 260 mM Tris-HCl and 65 mM MgCl2 at pH ∼9.5, 1.0 µL of NucleixPlus Terminator Sequencing Blend containing 6 mM dATP, 6 mM dCTP, 6 mM dTTP, and 18 mM dITP, 1.0 µL each of dye-labeled terminators with original concentrations of 15 µM ddATP, 450 µM ddCTP, 4 µM ddGTP, and 900 µM ddTTP diluted with deionized water by factors of 17, 56, 9, and 83, respectively, 2.0 µL of 5 units/µL diluted Thermo Sequenase enzyme solution, which was made from 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) and Thermo Sequenase dilution buffer (10 mM Tris-HCl, pH 8.0, 1 mM 2-mercaptoethanol, 0.5% Tween-20, 0.5% Nonidet P-40), 2.0 µL of 2.5 mg/µL BSA solution, and 5.15 µL of autoclaved and deionized water. The total reaction volume was made up to 20 µL. Except for the dideoxy dye-terminator kit (Catalog No. 401095) obtained from ABI and 2.5 mg/mL BSA buffer received from Idaho Technology, all other chemicals for preparing the cycle-sequencing reaction mixture were purchased from Amersham. The reaction mixture for AmpliTaq FS polymerase was modified by adding 2 µL of 2.5 mg/µL BSA buffer to the identical components used in the ABI dye-terminator cycle-sequencing protocol. Both ds-DNA (100 ng of pGEM) and ss-DNA (250 ng of M13mp18) templates were tested. The temperature protocol for the Thermo Sequenase cyclesequencing reaction was adjusted to the following: the sample mixture was heated to 95 °C and held for 3 min; 30 thermal cycles were performed with denaturation at 96 °C for 2 s, annealing at 45 °C for 5 s, and extension at 60 °C for 3 min; then the sample was ramped to 95 °C and held for 3 min. The temperature ramp rate was set at its maximum value, 9.9 °C/s. When AmpliTaq FS polymerase was used, the step of 30 cycles was implemented with the following profiles: denaturation at 96 °C for 2 s, annealing at 50 °C for 10 s, and extension at 60 °C for 4 min. Also, standard pGEM/U and M13mp18/U DNA samples cycle-sequenced by AmpliTaq FS polymerase were obtained from the Nucleic Acid Facilities at Iowa State University (Ames, IA) for comparison. For off-line experiments, 10 µL of the reaction mixture was sealed in a 0.53-mm-i.d. soft-glass capillary by a microflame torch as described in the hot-air thermal cycler manual. The cyclesequencing reaction products were purified by the spin column obtained from Princeton Separation, Inc. (Adelphia, NJ). The effluent from the spin column was mixed, without centrifugation and drying, with 4 µL of 20% formamide containing 50 mM EDTA and then denatured at 95 °C for 4 min. Further dilution of the denatured DNA sample was also tested. A 10-20-min prerun of electrophoresis was used to stabilize the separation matrix. Two electrokinetic injection procedures were implemented for introducing the DNA sample into the separation capillary. For cold injection, the DNA sample after denaturation was placed on an ice bath for 2 min and then injected immediately. For hot injection, the eluted material from the spin column was denatured without adding the formamide-EDTA solution at 95 °C for 4 min, rapidly taken into a hot water bath or a heating block held at 65(17) Wittwer, C. T.; Garling, D. J. BioTechniques 1991, 10, 76-83.

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75 °C, and then immediately injected. Both injection procedures were performed at 6 kV for 20 s. Detection for off-line experiments was done at the blue channel (488-nm excitation, 530-nm longpass emission) as described below. Column Packing Procedure. The purification columns were prepared from 31-cm-long, 0.04-in.-i.d., 1/16-in.-o.d. PEEK tubing (Alltech, Deerfield, IL). Before the column was packed, the inner bore of the tubing was rinsed thoroughly with HPLC-grade methanol, deionized water, and then elution buffer. The ends of the column were cut cleanly without burr and distortion. A PEEK universal column adapter (Alltech) was used to connect the 1/16in.-o.d. PEEK column to a PEEK metal-free in-line filter (Alltech) with a 2-µm PAT (PEEK alloyed with Teflon) frit. A slurry consisting of 3 mL of elution buffer and 0.5 g of packing material was prepared and allowed to swell for 3-4 h at room temperature. 1× TE buffer solution was used as the elution buffer. The packing material was superfine G-25-50 Sephadex particles (Supelco, Bellefonte, PA). The spherical particles have 20-70 µm of swollen bead diameter with useful fractionation range from 1000 to 5000 MW for globular molecules, which should retain dye-labeled dideoxyribonucleotides but exclude the other longer DNA fragments. After full swelling, the bottom portion of the slurry was transferred to a clean stainless steel reservoir (1/4-in. i.d., 2-in. length) which was connected with the PEEK column filled with elution buffer. The reservoir and column were pressurized to 20 psi using a µLC-500 pump (Isco, Lincoln, NE) with the degassed elution buffer. The PEEK column was allowed to fill with Sephadex particles and maintained at 20 psi for 1-2 h. The pump was then turned off, and the pressure was allowed to slowly bleed out over 1 h. A 1-cm piece of the column was trimmed off after removal from the reservoir. The column was bent to form a U-shape and inserted into the 25-50-µL capillary holder of the Rapidcycler before reconnecting with a PEEK metalfree in-line filter. When the capillary holder was placed onto the top of an air compartment of the Rapidcycler, the U-shaped part of the column was set into the compartment and the in-line filters and frits were isolated outside to prevent them from clogging at the relatively high temperature. The actual column bed is ∼250 µL and ∼30 cm long. Instrumentation and Operation. Figure 1 is a schematic of the entire instrumental setup. Two six-position valves A and B (Alltech), one switching valve C (Alltech), and another sixposition valve D were set on top of the Rapidcycler. Microreactor System. The microreactor MR was a 60-cm-long, 250-µm-i.d., 360-µm-o.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ). The capillary with ∼30 µL volume was placed inside the front capillary compartment, forming several loops to fit. The two ends of the capillary were connected to the center ports of valves A and B just above the compartment. Each end of the fused-silica loop was held in place in the valve by sleeving through Teflon tubing “liners”. The liners were made from 2-cm lengths of 0.015-in.-i.d., 1/16-in.-o.d. Teflon tubing (Valco Instruments, Houston, TX). Each of these liners has a standard ferrule and a standard 1/16-in. stainless steel nut for fittings. Care must be taken to avoid leaking and crushing of the capillary by the fittings when the nuts are installed into the valves. Cleaning of the capillary microreactor was performed by selecting different ports of the two valves. Syringes (5 mL) were connected to the ports through 25-cm-long pieces of 0.02-in.-i.d., 1/ -in.-o.d. Teflon tubing. Syringes S1, S2, S3, and S5 contain 1× 16 666

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TE buffer, HPLC-grade methanol, deionized water, and 0.2 M NaOH, respectively. After the microreactor was treated with reagents, helium gas (Air Products) was used to blow out the deionized water. A 2-µm-thick in-line filter unit AF (Alltech) removed particles from the gas and prevented sudden changes in pressure when the gas valve was opened. Teflon tubings (0.04in. i.d., 1/16-in. o.d.) (Valco) were used to link the junction T with AF, port A5, and port B4. The distance from A5 to T has to be exactly equivalent to that from B4 to T in order to achieve equal pressure. Before loading the samples, the transfer line T1 was cleaned by exactly the same procedure as described for the microreactor. The cycle-sequencing reaction mixture was loaded into the capillary microreactor at port B3 by vacuum produced by a 100µL glass syringe S4 (Hamilton) at port A6. A 10-cm-long, 150µm-i.d., 360-µm-o.d. fused-silica capillary (Polymicro Technologies) with ∼1.8 µL of dead volume was connected to port B3, and a 15-cm Teflon tubing with 0.02-in. i.d. connected the glass syringe to port A6. The six-position valve has ∼0.7 µL of dead volume. Therefore, by dipping the clean tip of the loading capillary B3 into the mixture vial and pulling the plunger of the glass syringe to 28 µL, the plug of reaction mixture solution will be placed in the center of the microreactor. Before temperature programming began, the microreactor was pressurized under 20-30 psi He by selecting ports A5 and B4. SEC Chromatographic System. The purification column PC was connected between port C4 and the center port of valve D via a 150-µm-i.d. fused-silica capillary (as short as possible). A µLC500 pump (Isco) was used to deliver an elution buffer to the column. The buffer was allowed to flow at 10 µL/min for 2 h before the column was tested. Two transfer lines (T1, T2) were installed. The one from port B5 to port C3, T1, was made from a 15-30-cm-long, 150-µm-i.d. fused-silica capillary while the one from port D6 to cross K, T2, was a ∼70-cm-long, 75-µm-i.d., 360µm-o.d. fused-silica capillary with a window ∼25 cm away from cross K. All waste lines (W1-5) were made from Teflon tubing, except that W2 was a 10-cm-long, 150-µm-i.d. fused-silica capillary linked to port C2. After the sequencing reaction was complete, chromatographic injection via transfer line T1 was accomplished by pressurizing the products at 30 psi for 1 min at port A5. Note that the gas in the transfer line needs to be swept out first. Therefore, the end of the waste line W2 was carefully monitored after port B5 was selected. Once a drop emerged at the tip, valve C should be switched to the injection position. Also, different portions of the sample can be injected by timing the interval between the two switches. CE Electrophoretic System and Interface. A PEEK cross (Alltech) K connected the transfer line T2 with a polymer-filled separation capillary (SC) in the opposite direction, with the other two arms forming a flow channel. One of the arms was connected to one of three syringes P1-3 via a needle valve E by using Teflon tubing (0.02-in. i.d., 1/16-in. o.d.). Syringes P1-3 contained 5 mg/ mL BSA aqueous solution, deionized water, and 1× TBE buffer, respectively. The 1× TBE buffer syringe served as the electrophoresis buffer reservoir. A flow rate of 10 µL/min was provide by a syringe pump (KD Scientifics, Wood Dale, IL) during DNA separation. The other arm G was installed with a 10-cm-long, 0.02in.-i.d., 1/16-in.-o.d. stainless steel tubing, which serves as both the grounded electrode (cathode) for the CE system and a waste line

Figure 1. Schematic of instrumental setup for integrated on-line cycle-sequencing-SEC-CE system: A, B, and D, six-position selection valves; C, dual-position switching valve; E, needle valve; S1, 1× TE buffer; S2, methanol; S3, deionized water; S4, dry and clean syringe; S5, 0.2 M NaOH; W1-5, waste outlets; AF, in-line filter unit; T, PEEK tee; MR, microreactor capillary; T1 and T2, transfer capillaries; PC, purification column; TC, hot air thermal cycler; P1, 5 mg/mL BSA solution; P2, deionized water; P3, 1× TBE buffer; K, PEEK cross; HB, hot-water bath; G, grounded stainless steel tubing; SC, separation capillary; L1 and L2, lenses; MO1 and MO2, microscope objectives; IF, 500-nm long-pass filter; BS, beam splitter; LPa, 2 × 530 nm long-pass filters; LPb, 2 × 610 nm long-pass filters; LPc, 610-nm long-pass filter; PMT-a, -b, -c, photomultiplier tubes; A/D, DT2802 A/D interface.

from the purification column and the TBE reservoir. A voltmeter across a 1-MΩ resistor was placed between the conductive tubing and the true ground electrode to monitor the current. The whole junction was immersed in a grounded hot water bath (HB, Grant Instruments, Barrington, Cambridge, MA) held at 65-75 °C, but

the outlet of the stainless steel tubing was kept away from the water bath to avoid forming a short circuit. The separation capillary was installed after being filled with PEO matrix. It is important for the transfer capillary T2 and the separation capillary to extend ∼2 mm beyond their Teflon liners so that they can be Analytical Chemistry, Vol. 69, No. 4, February 15, 1997

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held near the center of the cross. A positive high-voltage power supply (Glassman High Voltage, Whitehorse Station, NJ) was used to drive the electrophoresis from the anode. Detection. A 1-mW 543.5-nm He-Ne laser (Melles Griot, Irvine, CA) was used for monitoring the material eluted from the purification column. An uncoated plano-convex lens L1 (Edmund Scientific, Barrington, NJ) with 12-mm focal length was used to focus the laser to the capillary window. A 10× microscope objective MO1 (Edmund) was used to collect the fluorescence perpendicular to the excitation laser. A RG610 cutoff filter LPc was employed to block the scattered light. A photomultiplier tube PMT-c (Products for Research, Danvers, MA) operating at 1200 V was used to generate an electrical signal. This constitutes detection channel C. An air-cooled Ar+ laser (Uniphase, San Jose, CA, Model 2213-150ML) with multiline emission was used for CE detection. The 488-nm line (4 mW) was separated out with an uncoated 60° glass prism (Edmund). The excitation lens L2 and collection objective MO2 were similar to the setup for monitoring the purification process. A 500-nm long-pass interference filter IF (Optosigma, Santa Anna, CA) was used to reduce stray light. A 50/50 beam splitter BS (Optosigma) was used to split the fluorescence into two channels for simultaneous monitoring by two photomultiplier tubes PMT-a and -b (R928, Hamamatsu Corp., Bridgewater, NJ) operated at 1000 V. The blue channel was formed by two RG530 long-pass filters LPa, and the red channel by two RG610 long-pass filters LPb. The photocurrents from the three channels were transferred directly through 10-kΩ resistors to a 24-bit A/D interface at 4 Hz (Justice Innovation, Palo Alto, CA) and stored in an IBM PC/AT computer (IBM, Boca Raton, FL). Before electrokinetic injection, the cross region was rinsed with at least 1 mL of BSA solution, followed by the same amount of deionized water. When PMT-c indicated that the first peak from the size-exclusion column reached the cross junction, injection was initiated at 6 kV for 30 s at the leading edge of this peak. The transverse flow stream was completely stopped by needle valve E during injection. After injection, the effluent from the purification column was directed to the waste line W5 so that the back pressure from the transfer line was released immediately. The needle valve E was reopened and a total of 0.5-1 mL of 1× TBE buffer solution was allowed to flush slowly through the cross junction before electrophoretic separation. An electric field strength of 230 V/cm was typically used in all the experiments. Base Calling. A half-frame shift between the blue and red channels (due to asynchronous operation of the A/D converter) was first corrected by software (GRAMS/386, Galactic Industries, Salem, NH). A base-calling program recently developed in our laboratory18 based on the two-color ratio19 was used. Manual base calling was also conducted after using software to correct the baseline of the electropherograms.

A lofty goal in the Human Genome Project is to design an instrument that can perform DNA sequencing in an on-line format, starting from template, through sequencing reaction, on-line purification, on-line denaturation, on-line injection, capillary electrophoretic separation, and ending with called bases. Such a

system will be compatible with a high degree of multiplexing. The important first step is to show that an integrated on-line system is feasible. Here we study the critical parameters in each of the many steps from template to called bases. An optimized combination is demonstrated, albeit in a manual mode of operation and with bulky switching valves and connectors. Future development of this scheme is envisioned to make it fully automated and highly multiplexed through miniaturization. Sequencing Microreactor. One of the potential problems is that a large surface-to-volume ratio can inhibit DNA amplification due to adsorption. At normal cycle-sequencing conditions, the pH of the reaction mixture is typically above 8. DNA template, dNTPs, ddNTPs, and products of the reaction are therefore not expected to be adsorbed on the surface due to charge repulsion. However, polymerases are susceptible to denaturation via interaction with the negative surface. To recover the activity of these polymerases, BSA has been found effective in inhibiting adsorption.20 Preferential adsorption of BSA onto the capillary wall protects the polymerase from denaturation. Studies have not found that BSA has any negative effects on PCR or cycle sequencing. It is interesting to note that it is not necessary to add BSA when Thermo Sequenase was used in the reaction, but BSA must be added when AmpliTaq FS and the ABI kit were used. This may be the result of the large portion of glycerol that exists in the Thermo Sequenase storage solution or neutral surfactants such as Tween-20 and Nonidet P-40 in the dilution buffer. It is also possible that Thermo Sequenase can tolerate surface denaturation better than AmpliTaq FS. To perform cycle sequencing in a glass capillary requires a vapor-tight system. If the capillary is used with the ends open, a serious problem observed is the segmentation of the solution plug inside the capillary due to nonuniform heating. When a fusedsilica capillary is used to replace the soft-glass capillary, it is very hard to seal and reopen the capillary with flames due to the high melting point of fused silica. Besides, sealed capillaries are not compatible with on-line operation. We found that pressurization of the capillary microreactor under 20-30 psi effectively suppressed segmentation of the solution plug. Standard DNA templates of pGEM for ds-DNA and M13mp18 for ss-DNA have been successfully used for cycle sequencing in this manner. Figure 2a shows the electropherogram of M13mp18 DNA template cycle-sequenced by Thermo Sequenase in a pressurized fusedsilica capillary. Both Thermo Sequenase and AmpliTaq FS have been used successfully. The intensity and peak pattern in the electropherograms for samples obtained from the pressurized reactor are similar to those of the sealed capillary reactor. An additional advantage in using a pressurized reaction chamber is that the He gas can also be used as the driving force in transferring and injecting the Sanger reaction products into an on-line purification column. To eliminate cross-contamination between runs, a cleaning procedure has been developed. The capillary microreactor MR, loading capillary at port B3, and transfer capillary T1 must be flushed with 0.2 M NaOH solution between runs. This helps to remove interference for the next run through deprotonation of the fused-silica surface and denaturation of DNA fragments and proteins. Subsequent washing with 1× TE buffer favors the removal of DNA. Methanol further dissolves all hydrophobic

(18) Hazen, K.; Yeung, E. S., unpublished results. (19) Li, Q.; Yeung, E. S. Appl. Spectrosc. 1995, 49, 1528-1533.

(20) Wittwer, C. T.; Fillmore, G. C.; Hillyard, D. R. Nucleic Acids Res. 1989, 17, 4353-4357.

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Figure 2. (a) Electrophoretic separation of DNA fragments after cycle-sequencing reaction for M13mp18/U ss-DNA templates amplified by Thermo Sequenase in a fused-silica capillary microreactor. (b) Electropherogram for a blank cycle-sequencing reaction (without DNA template) under the same conditions as (a) and in the same capillary after extensive flushing. Both electropherograms are plotted on the same scale.

ingredients. Then, a large amount of deionized water is used to flush the residues out under relatively high pressure. Compressed He gas finally removes all water and dries both the reactor and the transfer capillaries. The whole operation was done at high temperature, except for the NaOH wash. At least 1 mL of reagent was used in each step. After a standard cycle-sequencing reaction followed by this cleaning process, a sequencing reaction mixture without DNA template was reloaded into the capillary microreactor. The reaction products, purified by either a spin column or homemade Sephadex column, were then injected into a separation capillary to check for cross-contamination. A typical electropherogram of these purified samples is shown in Figure 2b. In this example, interference to the sequencing run, albeit minor, occurs within a few minutes after the first eluted peak. In the actual on-line system discussed below, we did not observe even the small interference peaks as a result of the longer purification column and the smaller residual amounts of dye terminators. This suggests that the cleaning procedure does effectively remove all interference from the previous reaction. This shows that it is possible to reuse the microreactor, which is essential for multiplexed high-throughput operation envisioned for the Human Genome Project. Loading the sequencing reactants into a capillary by a microaspirator at valve A is straightforward. Dipping the tip of the loading capillary into the bottom of a vial in a 96-well microtiter plate, and driving the reaction mixture into the reaction loop is also very easy. As little as 1 µL of solution can be taken up by the combination of aspiration and capillary action.

Purification. Injection from the capillary microreactor to the size-exclusion column was performed by pressurizing the reaction mixture via valve A at 20-30 psi for ∼1 min. The gas in the transfer capillary T1 was first swept out through waste line W2 and then replaced with the reaction products. Careful manipulation of the time interval between selecting valves B and C will assure that an appropriate plug of sample is sent into the purification column. The interval between the switching times is typically less than 1 s. Fortunately, reproducibility of injection here is not important as long as the purification column can effectively remove the fluorescently labeled terminators. With future implementation of automated valves, it should be possible not only to obtain high reproducibility but also to further optimize the switching interval between valves B and C. No significant difference was found whether the transfer capillary was placed inside the thermal cycler or outside. This indicates that renaturing of the sequencing products is not important in the transfer capillary. However, it is convenient here to have the transfer capillary inside the thermal cycler. Also, it is more effective to clean and regenerate the column under high temperatures. To study the purification process, we added 4 µL of formamide-EDTA solution to the reaction products, followed by denaturation, and then placed the mixture in Teflon tubing connected to a microsyringe (on an ice bath). After injection (∼5 µL) into the transfer capillary T1, the sample was allowed to pass into a 30-cm Sephadex column. This was eluted at 10 µL/min by 1/100× TBE buffer or deionized water and monitored in detection Analytical Chemistry, Vol. 69, No. 4, February 15, 1997

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Figure 3. Purification of crude cycle-sequencing reaction products at different temperatures with 10 µL/min flow rate in a 30-cm 0.04in.-i.d. Sephadex G-25-50 column: (a) at 0 °C with 1/100× TBE buffer, ∼5 µL injection; (b) at 95 °C with 1/100× TBE buffer, ∼5 µL injection; (c) at 95 °C with 1× TE buffer, on-line injection by 20 psi for 1 min. Thermo Sequenase cycle-sequencing reactions were done in soft-glass capillaries except for (c), which was done in a fusedsilica capillary.

channel C, the signal from which is shown in Figure 3a. The first peak is the DNA fragment nested set, and the second peak is the labeled ddNTPs. Incomplete separation was obtained at low temperature (0 °C) over the entire range of column capacity (5-50-cm effective packing length) and flow rate (5-50 µL/min). On the other hand, when we placed the Sephadex column inside the hot-air thermal cycler held at 95 °C during separation, we found that the two peaks (Figure 3b) were easily resolved, even without the addition of the formamide-EDTA solution. The tailing in Figure 3b is probably due to poor packing of this column, which lowers the performance of the separation. To test the purity of each peak, the fractions of the peaks in both low- and high-temperature elution were collected and analyzed by CE. It is interesting that the fractions at the leading edge of the first peak from both low- and high-temperature experiments were still contaminated with material from the second peak, mainly labeled ddNTPs. This indicates that resolving the nested fragment set from the labeled ddNTPs in a low ionic strength solution cannot be achieved. Temperature should not affect the elution volume in SEC.21,22 In low ionic strength solutions, aromatic or ionic interactions are expected to play an important role in these separations. By replacing the 1/100× TBE buffer with a 1× TE buffer to increase the ionic strength (Figure 3c), it is clear that one can obtain baseline-resolved separation and improved peak symmetry for both peaks. In Figure 3c, retardation of the ddNTPs peak can be explained by the availability (21) Casassa, E. F.; Tagami, Y. Macromolecules 1969, 2, 14-17. (22) Adams, J. R.; Bickin, M. K. L. Anal. Chem. 1985, 57, 2844-2849.

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of the inner pore volume due to suppression of the negatively charged carboxyl groups in Sephadex. The slight decrease in the elution volume for the first peak is attributed to the suppression of aromatic interactions of purines and pyrimidines with Sephadex. The peaks in Figure 3c were collected and injected separately into the CE column. The overlap between the first peak and the second peak clearly disappeared. The drawback of using 1× TE buffer is the loss of the benefit of preconcentration by stacking during injection of the Sanger products later on. An increase in flow rate and decrease in column length are possible, as suggested by Figure 3c. Sephadex G-25-150, G-25-80, G-25-50, and G-50-50 (Supelco) and even spin column resin (Princeton Separation) were evaluated. It was found Sephadex G-25-50 is the best material to purify this set of dye-labeled terminators. Recycling a purification column is not often done in a molecular biology laboratory. However, this will be an important issue in a multiplexed, high-throughput operation. In this design, the purification column was placed in a hot-air thermal cycler, which promotes self-cleaning. A typical terminator-labeled cyclesequencing reaction is done in ∼2.5 h, and microreactor regeneration, sample loading, and filling of the separation matrix require ∼0.5 h. The 3-h, 10 µL/min flow gives ∼1800 µL of eluent, corresponding to six packed-column volumes. So, the column can be flushed during the sequencing reaction. This should be sufficient to remove the retained materials during purification. Figure S1a (Supporting Information) shows the electropherogram of the purification column eluent after 3 h of flushing with 1× TE buffer. There are a few interference peaks at the front end of the electropherogram. The signal magnitudes are however substantially lower than those in a sequencing run (Figure 2a). In comparison with the electropherogram in a prerun of the separation capillary (Figure S1b), the background-to-noise (S/N) ratio is comparable. So, carryover in the purification column is not a problem. Injection. By examining the peak widths in Figure 3, one can conclude that the sample plug from the microreactor was diluted by at least a factor of 10 during purification based on the peak elution volume of ∼50 µL. Also, typical DNA sequencing samples are obtained in dry form and are redissolved in a 4-µL formamide-EDTA solution before denaturation and injection. If instead the product mixture was used directly for injection (without preconcentration), another dilution factor of 5 exists because the standard cycle-sequencing reactions produce a total volume of 20 µL. So, the net dilution factor is ∼50. Therefore, a concern is how to inject a detectable quantity of diluted DNA sample into CE. To study the on-line injection process, standard dry pGEM/U and M13mp18 samples were diluted with deionized water, 1/100× TBE buffer, and 1× TE buffer, respectively, to mimic the matrix composition of the effluent from the purification column. A series of off-line CE experiments on these diluted samples were performed according to the cold injection method described above. Surprisingly, we found that DNA samples diluted by the low ionic strength solutions can tolerate up to 100-fold dilution without noticeable changes in S/N ratios and separation resolution when the injection voltage or injection time was increased. However, the samples diluted with 1× TE buffer (which has higher ionic strength) only allowed up to 20-fold dilution, producing a decrease

in S/N ratio by a factor of 5. The effect of the low ionic strength buffer on injection is presumably attributed to electric field amplification.23-25 No sampling biases have been found in free solution electrophoresis because DNA fragments have equal mobilities.26 Sampling bias does occur in gel matrices if electroosmotic flow is present.27 Here, no sampling bias was observed. With the promising results related to injection of diluted DNA samples, we modified the transverse-flow gating design for SECCE28 for sample injection. Here, a simple PEEK cross from standard HPLC hardware was used for connection. To test the on-line injection scheme, the transverse flow stream of the cross junction was connected with a 5-cm-long, 75-µm-i.d., 360-µm-o.d. fused-silica capillary by a Teflon sleeve. A 12-cm-long, 0.015-in.i.d., 1/16-in.-o.d. Teflon tubing was then used to join the capillary to a 10-µL microsyringe. Either a pGEM/U or a M13mp18 DNA sequencing sample was purified and denatured in a manner similar to the standard off-line, cold injection method. The whole cross and microsyringe with their connection tubings were immersed in an ice bath to prevent the DNA from renaturation. Initially, by slowly pushing the plunger during electrokinetic injection, the DNA sample was delivered to the cross junction. However, not much signal was observed. After rinsing the cross region just before injection with cold deionized water, we were able to restore the signal level, but a large, reproducible baseline fluctuation was observed. The baseline always goes back to its original level toward the end of the separation runs. This indicates that adsorption of the sample in the cross and the tubings is the problem. The preferential adsorption of BSA in a fused-silica capillary has previously been used to prevent adsorption of the polymerase. So, ∼1 mL of 5 mg/mL fresh BSA solution is utilized to flush through the junction, followed by deionized water. The cold denatured samples are introduced into the cold cross region. After injection, TBE buffer is used to slowly wash out everything in the stream. For manual operation, some care needs to be taken for slowly flushing TBE buffer into the junction. It is not desirable to leave the sample plug in contact with the separation matrix for a long period due to diffusional broadening. However, it is also not desirable to flush the junction so fast that rapid flow near the interface disturbs the injection plug. After the junction is cleaned, electrophoretic separation is initiated while 1× TBE buffer is kept flowing at 10 µL/min past the junction. Denaturation. Chemicals, salts, and heat are the common agents for denaturing DNA. In standard DNA sequencing protocols, the purified dry sequencing products are heated in a denaturing solution at 95 °C for a few minutes and then loaded into the slab gel. However, on-line denaturation may not be necessary because the purification column is operated at very high temperature and because of relatively fast sample transfer. To test this hypothesis, a separate experiment was conducted by placing the purified DNA sequencing sample in the microreactor and reconnecting the transfer line T1 to the cross junction which (23) Chien, D. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (24) Guttman, A.; Schwartz, H. E. Anal. Chem. 1995, 67, 2279-2283. (25) Butler, J. M.; McCord, B. R.; Jung, J. M.; Wilson, M. R.; Budowle, B.; Allen, P. O. J. Chromatogr., B 1994, 658, 271-280. (26) Goodall, D. M.; Williams, S. J.; Lloyd, D. K. Trends Anal. Chem. 1991, 10, 272-279. (27) Klepamik, K.; Garner, M.; Bocˇek, P. J. Chromatogr., A 1995, 698, 375383. (28) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581.

was held at 0 °C. After denaturation at 95 °C, a high pressure of He gas was used to send the DNA sample into the cross junction in ∼2 s. The result is band compression and loss of resolution for the later migrating DNA fragments in the electropherograms. Kinetic studies29 suggest that renaturation of DNA depends on denaturing and renaturing temperatures, ionic strength, pH, salt identity, surfactants and their concentrations in the sample buffer, and sample heat capacity. In the present system, the transfer line T2 has a volume of ∼8 µL, which requires ∼1 min of flushing time. The renaturation rate of DNA is so fast that it is not possible to preserve the denatured DNAs. Adding a denaturing reagent at a postcolumn reactor will not only complicate the flow control system but also further dilute the sample plug. The use of high temperature to denature DNA is very common in PCR. This led to the implementation of “hot injection” to guarantee that the fragments remain denatured. The whole cross junction was placed in a heated water bath. The purified sample was sent from the transfer capillary T2 into the cross region without the addition of any formamide-EDTA solution. When the leading edge of the first peak (Figure 3) appeared in detection channel C, the injection voltage was then turned on, typically at 6 kV for 30 s. After the injection process, valve D must be switched to the waste line W5 to redirect the flow coming from the column. Careful inspection of the electropherograms (Figire S2) reveals there is very little difference between the two injection methods in terms of S/N ratios, peak patterns, efficiency, and resolution of separation. It is possible that the injected plug length is broadened by the increase in electrophoretic mobility and thermal diffusion at the high temperature. On the other hand, high temperature has been shown to increase the resolving power for longer DNA fragments. Here, after injection, the cross junction was removed from the water bath to prevent nonuniform local heating and bubble formation. To optimize the injection temperature, the purified DNA sample without the addition of denaturing reagents was heated at 95 °C for 4 min and rapidly transferred to a hot-water bath held at different temperatures. It was found that the optimal temperature for denaturation of pGEM or M13mp18 DNA is 65-75 °C. If the temperature is too low, the DNA sample does not completely denature, but if the temperature is too high, bubbles form easily at the tip of the capillary. Hyperchromicity analysis,30 in which relative absorption vs temperature is measured, has shown that most DNA samples can be denatured in a solution with relatively low ionic strength at a temperature around 70 °C if the GC content of the DNA is below 60%. This alleviates the concern about the completeness of denaturation at this temperature range. However, limitations in sequencing DNA with high GC content may still exist. The reasons for the effective on-line injection in 1× TE buffer are unclear. The high solubility of DNA samples in this buffer should reduce possible adsorption loss. Continuous flow from the purification column during injection might have enhanced mass transfer. Elimination of potential complexation of borate with DNA might also be involved. Finally, the high temperature causes higher electrophoretic mobilities in free solution, which can in turn cause more material to be injected. (29) Wetmur, J. G.; Davidson, N. J. Mol. Biol. 1968, 31, 349-370. (30) Freifelder, D. In Physical Biochemistry, 2nd ed.; W. H. Freeman & Co.: New York, 1982; pp 507-510.

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Integration. It is shown above that when a low ionic strength buffer such as 1/100× TBE buffer is used, the poorer performance of the purification column causes interference in the electropherograms from fluorescently labeled ddNTPs. However, a low ionic strength benefits on-line injection and provides more effective sample stacking. We found that 1× TE buffer seemed to prohibit on-line injection at a cold junction. Surprisingly, the heated junction permitted on-line injection to CE with 1× TE buffer. This result removes the final barrier to complete integration of on-line DNA sequencing.

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Figure 4 shows the electropherograms from on-line cycle sequencing, purification, injection, and separation of a M13mp18 sample recorded by using one-wavelength excitation and dualwavelength detection.19 The fluorescence signal in the blue channel is reduced by a factor of 4 and offset by 2000 units in order to fit these on the same plot. Data in both channels show very high S/N ratios, without any interference from dye-labeled ddNTPs. Resolution factors at better than 0.5 can still be seen toward to the end of the separation. By using in-house software,18 we were able to call the sequence from 36 to 360 bp with an

Figure 4. On-line integrated sample preparation and electrophoretic separation of M13mp18 ss-DNA sequencing fragments by using one wavelength excitation and dual-wavelength detection: blue channel, top; red channel, bottom; loading of purification column, 1 min at 30 psi; injection for separation capillary, 35 s at 6 kV just before leading edge of the first peak in channel C reached its plateau; cycle-sequencing reaction was done with Thermo Sequenase; elution of purification column, 10 µL/min flow rate; applied electric field, 232 V/cm.

An on-line DNA sequencer from template to called bases with capillary electrophoresis has been demonstrated in a capillary format. The studies here help define the subtle features of each of the many steps critical to sample preparation and electro-

phoretic injection. The protocol for cycle-sequencing reaction and operational procedure have thus been optimized. Conditions very different from standard protocols were needed to assure compatibility throughout. Electropherograms obtained by using onewavelength, one-beam excitation, and dual-wavelength detection allow manually assisted base calling on the raw data up to 370 bp with 98% accuracy. A major advantage for the on-line operation of DNA sequencing is the potential for automation of the entire system. Computercontrolled valves and pumps are common techniques in the chromatographic world. To adapt to a multiplexed capillary array detection system, matrix fill/flush of the separation capillary can be done from the detection end by a pressure cell described previously.16 The major barrier for multiplexed operation of this on-line system is the use of four flow valves. Multiplexing 4 × 100 selection valves is quite clumsy. However, one just needs four computer-controlled valves and a distribution network to feed into each of 100 channels, since the protocol is identical in each. Also, the use of freeze-thaw control of liquid flow without valves has been demonstrated.32 Such a system allows the complete elimination of bulky valves and junctions. Still, to our knowledge, this is the first report of on-line coupling of the four-color cyclesequencing reaction to capillary electrophoresis, allowing base calling without any off-line manipulation. Future miniaturization and multiplexed operation are envisioned not only in DNA sequencing and mapping but also in genetic typing, population screening, and drug discovery.

(31) Parker, L. T.; Zakeri, H.; Deng, Q.; Spurgeon, S.; Kwok, P.-Y.; Nickerson, D. A. BioTechniques 1996, 21, 694-699.

(32) Bevan, C. D.; Mutton, I. M. Anal. Chem. 1995, 67, 1470-1473.

accuracy of 96.5% directly from the raw data. The majority of miscalled bases in several runs was “C” when Thermo Sequenase polymerase was used. Most errors occurred at “G” following “A” if AmpliTaq FS was employed. These are associated with known intensity problems with the protocols.19,31 The crude baseline correction algorithm resulted in negative values for some of the fluorescence signals in the blue channel so that a miscall often occurred in that region. If the very front part of the electropherogram was omitted, the in-house software gave a better baseline correction. By manually examining the ratiogram, the accuracy improved to 98% through 370 bp. Naturally, more sophisticated software, such as those used in commercial sequencing instruments, should alleviate such baseline problems. For three consecutive runs of M13mp18 ss-DNA template in the complete system, the retention time, signal intensity, and resolution of separation were comparable to the variations in the off-line separations. pGEM ds-DNA template was tested right after an on-line run for M13mp18 template. The correct sequence of pGEM DNA also can be called for the first 300 bases. This indicates that the carryover from run to run in the on-line system is negligible.

CONCLUSION

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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. SUPPORTING INFORMATION AVAILABLE Figures showing that cold injection and hot injection produced identical electropherograms for the Sanger fragments (S1) and

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that carryover is negligible in the purification column after washing (S2) (4 pages). Ordering information is given on any current masthead page. Received for review September 5, 1996. December 4, 1996.X

Accepted

AC960892E X

Abstract published in Advance ACS Abstracts, January 15, 1997.