Automated and Integrated System for High-Throughput DNA

An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capil...
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Anal. Chem. 1999, 71, 1138-1145

Automated and Integrated System for High-Throughput DNA Genotyping Directly from Blood Nanyan Zhang, Hongdong Tan, and Edward S. Yeung*

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

An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput. The widespread application of the polymerase chain reaction1 (PCR) has prompted the rapid development in integration of all of the steps of DNA assays in a single platform in order to achieve high speed, high throughput, low cost, and full automation. Toward this end, a hot-air thermal cycler coupled with capillary electrophoresis (CE)2,3 and a miniaturized thermal cycler integrated with microchips4 have been demonstrated for the purpose of coupling PCR to the DNA separation and analysis steps. However, these published results did not start with native biological samples and can only process one sample at a time. The suitability for large-scale operations is therefore still limited even though the individual steps can be automated and miniaturized. In the clinical and forensic laboratories, conventional PCR diagnosis, in most cases, starts from whole blood. The genomic (1) Templeton, M. S. Diagn. Mol. Pathol. 1993, 1, 58-72. (2) Zhang, N.; Yeung, E. S. J. Chromtogr., A 1997, 768, 135-141. (3) Swerdlow, H.; Jones, B. J.; Wittwer, C. T. Anal. Chem. 1997, 69, 848-855. (4) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086.

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DNA is usually extracted or purified by various methods such as phenol/chloroform extraction prior to PCR. When the genomic DNA is the analyte and inhibitors to PCR such as hemoglobin are present, certain purification steps to remove these components are usually included. However, this requirement complicates the instrumentation due to the use of centrifugation, as discussed before.5 The alternative solution is to develop a more tolerant PCR protocol to amplify the genomic DNA directly from whole blood. Such a PCR protocol has been suggested by Panaccio and Lew.6-8 Although that protocol was developed for the amplification of a single DNA locus, it opens the door for multiplex PCR under analogous conditions. Another obstacle to full integration is that the surface-to-volume ratio and the material used for the reaction vessel can also be considered as inhibitors. These were observed to affect PCR efficiency and specificity.9-11 The development of more tolerant PCR protocols in small inner diameter capillaries will ultimately provide the ability to analyze real biological samples on a large scale when reagent cost and instrument size are important considerations. To manipulate multiple samples simultaneously for PCR analysis, a controlled parallel microfluidics system needs to be developed to integrate with capillary array electrophoresis or microchip devices. Recently, our group has successfully demonstrated an on-line multiplexing scheme for automatic DNA sequencing from template to base calling in eight parallel channels.12 That scheme, which is based on liquid nitrogen freeze/ thaw valves and pressure-driven fluidics, is expandable to a large number of channels without much modification. In contrast to DNA sequencing applications, it is not necessary to purify PCR products for genotyping from blood. However, it is necessary to add standards for calibration to pinpoint matches.13 This complicates fluidic control. In this study, a protocol for simultaneous PCR for three shorttandem-repeat (STR) loci, vWA, THO1, and TPOX, directly from (5) Tan, H.; Yeung, E. S. Anal. Chem. 1997, 69, 664-674. (6) Panaccio, M.; Lew, A. Nucleic Acids Res. 1991, 19, 1151. (7) Panaccio, M.; Georgesz, M.; Lew, A. Biotechniques 1993, 14, 238-243. (8) Panaccio, M.; Lew., A. M. PCR Technology: Current Innovations; CRC Press Inc.: Boca Raton, FL, 1994; pp 151-157. (9) Wittwer, C. T.; Garling, D. J. BioTechniques 1991, 10, 76-83. (10) Shoffner, M. A.; Cheng, J.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 375-379. (11) Cheng, J.; Shoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 380-385. (12) Tan, H.; Yeung, E. S. Anal. Chem. 1998, 70, 4044-4053. (13) Zhang, N.; Yeung, E. S. Anal. Chem. 1996, 68, 2927-2931. 10.1021/ac981139j CCC: $18.00

© 1999 American Chemical Society Published on Web 02/05/1999

whole blood is demonstrated in fused-silica capillaries and integrated into capillary array electrophoresis with eight parallel channels. Simultaneously, sample and standard loading is incorporated into the instrument. The whole system is computer controlled, including steps for washing and regenerating the entire system after the run. There are no off-line or manual operations from blood to final genotype determination. The system can potentially be expanded to hundreds of capillaries to allow even higher throughput. EXPERIMENTAL SECTION Direct PCR from Blood. Blood Samples. Blood samples were collected from volunteers into heparinized and plain tubes. One microliter of each blood sample was mixed thoroughly with 15 µL of deionized formamide. Then the mixture was incubated at 95 °C for 5 min. After that, the mixture is ready for direct PCR. These blood/formamide mixtures were stored at room temperature. PCR Reagent Mixture. The PCR reagent mixture was prepared by combining (a) 5 µL of 10× polymerase reaction buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0 at 25 °C, and 1% Triton X-100; Promega, Madison, WI); (b) 5 µL of 10× dNTP mixture (2 mM each mixture; Idaho Technology Inc., Idaho Falls, ID); (c) 3 µL of 10× buffer with BSA and 40 mM MgCl2 (500 mM Tris, pH 8.3 and 2.5 mg/mL BSA; Idaho Technology); (d) 2.5 µL of 10× 2.5 mg/mL BSA (Idaho Technology); (e) 0.5 µL of T4 gene 32 protein (storage buffer: 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 50% glycerol (v/v), pH 8.0, 5 mg/mL; Boehringer Mannheim); (f) 1.5 µL of Tth DNA polymerase (Promega); and (g) 25 µL of deionized water. The working solution was prepared every day and stored on ice. The volumes can vary proportionately depending on the amount needed. PCR Protocol. The primers for each of the three short-tandemrepeat loci, vWA, THO1, and TPOX, were purchased from Promega. The total volume for each PCR solution is 20.0 µL. For single-locus PCR, the reaction mixture contains 14.0 µL of reagent mixture, 3.0 µL of blood/formamide mixture, 1.0 µL of the fluorescent primer pair solution, and 2.0 µL of deionized water. For triplex PCR, the reaction mixture contains 14.0 µL of reagent mixture, 3.0 µL of blood/formamide mixture, and 1.0 µL each of the three fluorescent primer pair solutions. The PCR reactors were 66-cm-long, 360-µm-o.d., 250-µm-i.d. fused-silica capillaries (Polymicro Technologies). This length was dictated by the physical placement of the commercial PCR unit. PCR was also tested in 150-µm-i.d. capillaries for miniaturization purposes. The programmable thermocycler was controlled by hot air (Idaho Technology). On-line PCR was performed with the following protocol: 2 cycles of denaturation at 80 °C for 1 min, annealing at 45 °C for 1 min, and extension at 60 °C for 1 min, followed by 25 cycles of denaturation at 80 °C for 1 s, extension at 45 °C for 1 min, and extension at 60 °C for 1 min. Standard Allelic Ladder Preparation. A CTTv STR Genetic Typing kit was purchased from Promega. The allelic ladder was diluted 600 times from its original concentration. Then the diluted ladder was used as a template for PCR amplification to produce the stock standard solution. PCR was performed in a 2400 ABI thermocycler according to the protocol provided in the kit. The stock allelic ladder solution obtained was highly concentrated. It was further diluted before use as explained below.

Instrumentation. Multiplexed Liquid Nitrogen Freeze/Thaw Valves. Eight capillaries were bundled together as the reactors for PCR. The bundle was threaded through a hole made in a copper block (see inset in Figure 1), which acts as an on/off freeze/thaw valve.12,14 Liquid nitrogen was controlled by a cryogenic valve (Valcor Scientific, Springfield, NJ), as shown in Figure 1, to turn the valves off. An air blower was used to thaw the ice plugs to turn the valves on. The temperature was sensed by a thermocouple which was placed close to the center of the copper block. There were two freeze/thaw valves (V1, V2) in the system (Figure 1) to control the flow in eight parallel channels. V1 actually acts simultaneously on both the entrance and exit ends of the capillary reactor. The temperature sensors were monitored by computer so that the cryogenic valves and the air blowers could be activated by the computer to control the state of the liquid handling valves. Fluid Control. The syringe pump (Kloehm Co., Inc., Las Vegas, NV) was a programmable precision liquid delivery system (Figure 2). It was equipped with a six-way distribution valve V6. The center port was connected to a 1-mL syringe. Two ports of V6 were connected to manifold M1 and manifold M2 to distribute solutions to a T assembly and a cross assembly, respectively. The other four ports were connected to separate reagent bottles with 0.1 M NaOH, MeOH, diionized (d.i.) H2O, and 1×TBE buffer. The connecting tubings were 0.03-in.-i.d., and 1/16-in.-o.d. Teflon tubes. Each of the eight T connectors had three limbs (Figure 2). One was the PCR reactor, which was a 66-cm-long, 360-µm-o.d., 250µm-i.d. fused-silica capillary. Another limb was connected through the manifold M1 to V6. The third limb was a 60-cm-long, 150-µmi.d., and 370-µm-o.d. fused-silica capillary for transferring the PCR products and standard ladder to the cross junction for injection. The four ends of each cross junction were connected to the transfer capillary, through manifold M2 to V6, through manifold M3 to waste, and the separation capillary, respectively. The cross assembly formed a multiplexed interface between the sample preparation system and the capillary array electrophoresis system. The design of a similar multiplexed interface was described by Tan and Yeung in detail.12 The functions of the cross junction included the following: (a) denaturation of DNA by raising the injection temperature; (b) electrical conductor (ground) for injection of the DNA sample; (c) electrical conductor (ground) for electrophoresis separation; and (d) maintenance of the buffer flow and collection of waste. DNA Separation and Detection. The separation capillaries were 80-cm-long, 76-µm-i.d., 365-µm-o.d. fused-silica columns. The effective lengths were 60 cm. The eight capillaries were flushed with methanol first and then filled with 2% of 1 000 000 Mn poly(vinylpyrrolidone) (PVP) in 1×TBE solution. This solution was shown to form a dynamic coating on the capillary walls so that electroosmotic flow was eliminated.15 Finally, the separation matrix, which was 1.5% Mn 8 000 000 PEO and 1.4% Mn 600 000 PEO mixture in 1×TBE with 3.5 M urea, was filled into each capillary. A high-voltage power supply (Spellman, Plainview, NY) was used to drive the DNA fragments to the detection end. The PEO polymer solution was pushed out after each run. The capillary bundle was then flushed with d.i. H2O before the next run. (14) Bevan, C. D.; Mutton, I. M. Anal. Chem. 1995, 67, 1470-1473. (15) Gao, Q.; Pang, H.; Yeung, E. S. Electrophoresis, in press.

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Figure 1. Schematic of multiplexed liquid nitrogen freeze/thaw valves and their arrangement in the experimental setup. The two valves serve as the shut-off valves in the PCR capillary bundle and transfer capillaries. The liquid nitrogen flow was controlled by cryogenic valve 4. Hot air from air blower 5 was used to open the valve. The inset shows the cross-sectional view and dimensions of the valve. Temperature was sensed by the thermocouple. Number 1 is the T assembly, and 2 is the cross assembly. Number 3 is the holder for simultaneous injection.

Detection windows (∼2 cm) were made by burning off the polyimide coating carefully. The window region of the capillaries were packed side by side and then clamped onto a capillary holder. Laser light (15 mW of 488 nm) from an air-cooled single-line argon ion laser (Uniphase, Palo Alto, CA) was passed through a 60° prism (Edmund Scientific) to separate the plasma emission. Then the beam was focused by a 40-mm focal length convex lens (Edmund Scientific) to the first capillary in the array. The laser beam was also adjusted to be coplanar with the capillary array with a 20° incident angle.12 The system was installed in a class IV laser laboratory. The beam is enclosed in a sheet-metal box during operation. Fluorescence from each capillary was collected simultaneously by a two-dimensional cooled CCD camera (Photometrics, Tucson, AZ) from a direction perpendicular to the plane of the capillary array. The CCD camera had a lens (Canon, 70-mm diameter and 24-mm focal length) attached. One holographic notch-plus filter at 488 nm (Kaiser Optical System, Ann Arbor, MI) was used between the lens and CCD head. A 520-nm edge filter (Ealing Electrooptics, Hollison, MA) was used in front of the lens. The number of pixels that covered each capillary were 7 × 3. The exposure time was 300 ms. Frames were converted into a raw data file and then the raw data file was extracted as eight ASCII files at the pixels corresponding to the centers of each capillary. Automation and Control. A Dell GXMT 5100 computer (Dell Computer Corp., Austin, TX) equipped with a multifunctional data acquisition board (National Instruments, Austin, TX) was used 1140 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999

as the control hardware. Labview (National Instruments) was the control software for programming. Two serial ports originating from the computer provided two communication channels through the RS-232 protocol. They were employed to control the syringe pump with its six-position valve and the temperature controller (Omega) at the cross assembly. The three analog input signals monitored by the multifunctional data acquisition board were the temperatures of the thermocouples in each of the two freeze/ thaw valves and the total current of electrophoresis. Lines of digital I/O were connected to the relays (ER/16, National Instruments) for activating/deactivating the cryogenic valves, air blowers, plus the heater and the cooling fan for the cross assembly. All experimental procedures were computer controlled except for gel loading and operation of the hot-air thermocycler. Operation. Sample Loading and PCR. The syringe pump was set at a continuous pumping mode with d.i. H2O as the inlet and M1 as the outlet on V6. Initially, V1 was open and V2 was closed. The bundle of capillary microreactors were thereby flushed with 3 mL of d.i. H2O and the capillaries should be filled with d.i. H2O after this operation. A 20.0-µL aliquot of each PCR mixture was put into eight wells in a row of a microtiter plate. The loading ends of the eight capillaries were held by a holder and dipped into the eight wells (Figure 1). A 18.0-µL sample of PCR mixture was loaded into each capillary by aspirating the syringe pump for 144 µL (8 × 18). Afterward, 9.0 µL of d.i. H2O was loaded by aspirating into each capillary from a second row of wells on the microtiter plate. The PCR mixture plug was thus put right inside

Figure 2. Schematic of the complete multiplexed and integrated instrumental design with eight channels. Different functional capillaries were connected by a T assembly and a cross assembly. Stars represent the freeze/thaw valves V1 and V2. A syringe pump and a six-way selection valve were used to load and distribute liquids for various purposes. A CCD camera was used to collect fluorescence from the eight capillaries simultaneously. The temperature control unit at the cross assembly was used to denature DNA prior to injection.

the capillary loop within the thermocycler. The loading speed was 50 µL/channel per min. The loading tips were always kept in the water-filled wells. The PCR process started after V1 was closed. When PCR was in progress, the separation capillaries were prepared as described in the DNA Separation and Detection section. It is important to avoid bubbles or drying of the gel-filled capillary tips when they are fitted into the cross assembly. Filling the cross assembly with water ahead of time should prevent this. Also, flushing the cross assembly with d.i. water before injection was helpful for stacking injection.12 The alignment of the laser and image focusing of the CCD could also be done during this period. Allelic Ladder Loading and Sample Injection. Figure 3 shows the flow management during these operations. After PCR, V1 was opened and V2 was closed. The standard allelic ladder was loaded into the capillaries by the following steps. First, the temperature at the cross assembly was raised to 90 °C to be ready for DNA denaturation. Second, 300 µL of d.i. H2O from port 3 of V6 was aspirated into the syringe. Then, the syringe pump was reversed to dispense 72 µL to M1 to push out the excess water inside the reaction capillaries at the loading end. The third step involved putting the loading tips into another row of wells in the microtiter plate, each of which were filled with 12 µL of standard allelic ladder solution. The syringe was reversed again to aspirate a total of 80 µL. This way, 10 µL of allelic ladder was loaded into each capillary right after the individual PCR plugs. By changing the tips to the water-filled wells once again and aspirating the syringe another 270 µL, one can pull all the PCR products and the allelic ladder

plugs into the Teflon tubes between M1 and the T assembly. Then, with V1 closed and V2 open, reversing the syringe pump resulted in pushing the samples toward the cross junctions. Electrokinetic injection was started 13 s after this last operation. Injection lasted ∼33 s. The aspirating and dispensing speed was kept at 50 µL/ channel per min. Immediately after injection, the syringe pump started to pump 1×TBE buffer to the cross assembly with a speed of 200 µL/channel per min. High voltage was turned on to begin separation. Both injection and electrophoresis were performed at 12 kV (150 V/cm). The temperature at the cross assembly was rapidly lowered by the cooling fan to room temperature. Cleaning and Regeneration. With V1 closed and V2 open, the transfer capillaries between the T and the cross assemblies could be cleaned by flushing with NaOH, H2O, methanol, and H2O sequentially. The cross was also washed in the process. This can be implemented during PCR reaction for the next set of samples. The capillary reactors were cleaned at 80 °C (by ramping the temperature of the thermocycler) with 5 mL each of NaOH, H2O, and methanol sequentially, followed by flushing with large amounts of H2O at room temperature while V1 was open and V2 was closed. This can be performed during electrophoretic separation. For storage overnight, the separation capillary bundle was flushed with d.i. H2O using an HPLC pump after the gel matrix was pushed out. RESULTS AND DISCUSSION Polymerase Chain Reaction. In developing triplex PCR for vWA, THO1, and TPOX loci directly from whole blood in a fusedAnalytical Chemistry, Vol. 71, No. 6, March 15, 1999

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Figure 3. On-line operational protocol for sample introduction, PCR reaction, standard ladder loading, and injection. The operation was controlled by two freeze/thaw valves and a reversible syringe pump. See text for detailed operation.

silica capillary, a combination of approaches was followed. These include the formamide low-temperature (FoLT) PCR protocol,7,8 direct PCR from solid tissues,16 and other PCR applications.9,17 There are five major technical issues that are critical to success. First, diluting the whole blood sample should reduce the level of interference from all inhibitors, although it also reduces the concentration of the target DNA. As long as the dilution factor does not fall below the minimum allowable target concentration that permits reliable amplification, adequate amounts of PCR products can be produced by increasing the number of cycles. Second, high-temperature incubation of the raw sample prior to PCR and hot-start PCR should release many heat-sensitive inhibitors and keep the target DNA in a readily accessible state. Third, choosing a more tolerant DNA polymerase for PCR should result in amplification under severe conditions. Thermostable DNA polymerases from Thermus thermophilus (Tth) and Thermus flavus (Tfl) were found to have higher resistance to many inhibitors compared to Taq polymerase.18 Tth DNA polymerase19-21 has the unique property of maintaining both DNA- and RNA-dependent polymerase activities in the presence of 2%-5% (v/v) of phenolsaturated buffer or up to 24% of formamide buffer. Fourth, adding carrier proteins such as bovine serum albumin (BSA) and singlestrand DNA binding protein such as T4 gene 32 protein (T4gp32) should enhance PCR amplification efficiency,17 suppress inhibition from many contaminants,22 prevent absorption of DNA poly(16) Panaccio, M.; Georgesz, M.; Hollywell, C.; Lew, A. Nucleic Acids Res. 1993, 21, 4656. (17) Rapley, R. Mol. Biotechnol. 1994, 2, 295-298. (18) Wiedbrauk, D. L.; Werner, J. C.; Drevon, A. M. J. Clin. Microbiol. 1995, 33, 2643-2646. (19) Katcher, H. L.; Schwartz, L. BioTechniques 1994, 16, 84-92. (20) Glukhov, A. I.; Grebennikova, T. V.; Kiselev, V. I.; Severin, E. S. Mol. Biol. (Moscow) 1995, 29, 942-949. (21) Grebennikova, T. V.; Glukhov, A. I.; Chistiakova, L. G.; Kiselev, V. I.; Severin, E. S. Mol. Biol. (Moscow) 1995, 29, 930-941.

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merase9 on the reaction vessel, and promote DNA strand transfer and displacement in the DNA synthesis reaction.23,24 Fifth, the addition of formamide in the PCR mixture should reduce the degree of protein coagulation and allow more DNA template to be accessible for amplification.7 Formamide also lowers the melting temperature of DNA. By applying all five technical concepts at the same time and by fine-tuning all of the parameters that affect PCR amplification, we were able to achieve triplex PCR amplification of vWA, THO1, and TPOX loci from whole blood in fused-silica capillaries. The individual critical components are discussed in the following. Enzyme and Carrier Proteins. This study confirms that Tth polymerase is less sensitive to the presence of blood components than Taq polymerase. Amplification was unsuccessful when Taq polymerase was employed for PCR directly from blood. A total of 2.5 units of Tth polymerase was used in 20-µL reaction solution, which was a slightly higher concentration (1.25×) than conventional PCR in polypropylene tubes. BSA was also added at a concentration of ∼220 µg/mL to prevent enzyme adsorption and dissociation at the capillary wall, as suggested by the previous study.12,25 A single-stranded binding protein, T4 gene 32 protein, must be added to the PCR mixture (0.82 µg in 20 µL of reaction mixture) to facilitate triplex PCR of vWA, THO1, and TPOX. This protein increases the accessibility of Tth polymerase to the target DNA.6 Single-locus PCR was successful without this binding protein, but the efficiency of triplex PCR was very low without it. Formamide Concentration. The dilution range of blood in formamide can vary from 1/10 to 1/25 for acceptable PCR performance. The final concentration of formamide in the PCR (22) Kreader, C. A. Appl. Environ Microbiol. 1996, 62, 1102-1106. (23) Kong, D.; Nossal, N. G.; Richardson, C. C. J. Biol. Chem. 1997, 272, 83808387. (24) Salinas, F.; Kodadek, T. Cell 1995, 82, 111-119. (25) Zhang, N.; Yeung, E. S. J. Chromatogr., B 1998, 714, 3-11.

Figure 4. Direct PCR from blood. (A-C) are the electropherograms of products from single-locus PCR. (A) is the vWA locus, (B) is the THO1 locus, and (C) is the TPOX locus. (D-F) show the triplex PCR of vWA, THO1, and TPOX. (D) is blood sample 1 amplified in a 250-µm-i.d. fused-silica capillary. (E) is blood sample 2 amplified in a 250-µm-i.d. fused-silica capillary. (F) is blood sample 1 in a 150-µm-i.d. fused-silica capillary.

mixture was however a very important parameter in determining the success of PCR. Final formamide concentrations of 15%-19% were tested. Single-locus PCR was achieved with high yields in the entire range as long as the thermocycler parameters were set appropriately depending on the formamide concentration. For example, a lower temperature had to be used with higher formamide concentration and vice versa. Figure 4A-C shows some results from single-locus PCR. The workable concentration range of formamide for triplex PCR was narrower, being at 15%16%. The optimal concentration was found to be lower than the concentration suggested by Panaccio and Lew.8 Thermocycler Parameters. The annealing temperature was the most important parameter when adjusting the thermocycler. A temperature of 45 °C was found to be the best in this hot-air thermocycler. The denaturation and extension temperatures were 80 and 60 °C, respectively. It was necessary to use two cycles of long denaturation to pretreat the DNA, similar to the principle of hot-start PCR. The total time of PCR was about 1 h 20 min. Capillary Diameter. Fused-silica or glass capillaries (700-360 µm o.d.) with various inner diameters, including 500, 250, 200, and 150 µm, were tested off-line as the reactor for PCR. There was no significant difference in performance with respect to the capillary inner diameter if the PCR mixture contains the T4 gene 32 protein. However, PCR did show a higher failure rate when the inner diameter decreased if there was no T4 gene 32 protein in the PCR mixture. The results of triplex PCR of vWA, TPOX, and THO1 in 250- and 150-µm capillaries are shown in Figure 4DF. Yet smaller inner diameter capillaries were not tested in this study. The observation here that PCR efficiency is independent of surface-to-volume ratio suggests that the PCR reactor can be further miniaturized by using capillaries with even smaller inner diameter without any negative effects on its efficiency. This feature should lead to substantial cost savings in large-scale screening through a reduction in the amounts of samples and reagents used.

Overall Performance. The performance of direct PCR from blood in a fused-silica capillary depends on many factors. For example, on testing 10 blood samples from different (unidentified) individuals, we found the performance varied among these samples. Three samples showed poor results such as a large number of interference peaks or no amplification for triplex PCR. This is probably due to the presence of unknown substances in blood that reduces the activity of the polymerase. Single-locus PCR always performed well for any of the CTTv loci because a high amplification efficiency is always achieved to mask out any interference peaks. The blood samples can be stored at 4 °C in tubes containing heparin and EDTA for a long time without dramatic effects on the PCR performance. Acceptable storage times of the blood/formamide solution was up to four months at room temperature in capped vials. Multiplexed Instrumentation. Tan and Yeung demonstrated a liquid nitrogen-based freeze/thaw valve in which eight capillaries were packed in parallel.12 In this study, the eight capillary reactors were bundled together and threaded into a hole with 1.5-mm diameter made in a 2.5 cm × 2.5 cm × 1 cm copper block. Liquid nitrogen was directed perpendicularly to the capillary bundle from another hole with 4-mm diameter until the temperature was brought down to -20 °C (Figure 1 inset). The response time was 15 s for valve closing. To open the valve, hot air provided homogeneous heating to the copper block until the temperature reached 20 °C. The copper block is a good heat conductor. However, due to the low power of the blower used here (40 W), the response time for opening the valve was ∼1 min. Improvement in speed can be made by using a higher power heater or focusing the hot air to a smaller spot. The advantages of this design are the ease of arranging the capillaries and the capacity for higher throughput. For 96 capillaries, one only has to make a square hole with 3.6 mm on each side. If PCR can be performed in 75µm-i.d., 150-µm-o.d. capillaries, a 4.7-mm square-shaped hole can Analytical Chemistry, Vol. 71, No. 6, March 15, 1999

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hold 1000 such capillaries. The additional heat capacity should be negligible compared to the copper block. So this freeze/thaw valve design is readily scalable to a large array with minor changes in instrument size. Because there was no need for purification of the PCR products, all connections between the T assembly and the cross assembly were open tubes in this study. One syringe pump could provide enough pressure for liquid transfer in even 1000 channels. No expensive high-pressure pump was necessary, in contrast to DNA sequencing where a higher pressure is needed for sample cleanup.12 Also, only one multiposition valve was needed for the entire operation. For multiplexed operation, flow uniformity was critical because all operations, especially sample injection, were simultaneous for all channels. The flow rates in the eight channels here were quite uniform because of the open tubular character. The capillary length was the determining factor because all other parameters such as the diameter of capillary, solution viscosity, and temperature were essentially identical in each channel. One only needed to keep the capillary and the Teflon tubing lengths identical in each channel. Also, every port of the T assembly and the cross assembly should be kept identical. The flow uniformity was verified by observation with a CCD camera above the transfer capillary bundle near the cross assembly. Operational Protocol. Figure 3 shows the individual steps of sample introduction, PCR reaction, standard ladder loading, and injection. To put the PCR mixture and the allelic ladder in the appropriate regions of the capillary tubes, the volumes of aspiration and dispensing from the syringe must be calculated from the capillary length and inner diameter. The time delay for initiating electrokinetic injection (13 s) was calculated from the syringe flow rate and the transfer capillary volume. The injection duration (33 s) was obtained by dividing the total volume (28 µL) of the PCR mixture and the standard ladder solution by the flow rate of the syringe pump (50 µL/channel per min). The actual liquid handling performance was found to be very accurate based on these calculations. Each channel was always filled with H2O or other solutions during the whole process to prevent bubble formation at the cross junction. The standard ladder was loaded as a plug following the plug of PCR mixture. The two plugs were pushed to the cross junction and then injected one after the other. This method simplified the instrument design and was efficient because no additional channels were attached for standard loading. Repeated injection of the standard ladder over many runs from the same set of vials was possible. This reduces the amount and thus the cost of standard solutions needed. Previous studies15,26 showed that co-injection of PCR products and the allelic ladder (as an absolute standard) provides reliable identification of unknown alleles over a wide range of relative peak intensities. An intensity ratio between 0.5 and 1.5 is recommended.15 This can be achieved by adjusting the volume of the solution plug or the concentration of the allelic ladder solution. The injection temperature was set at 90 °C; 80 °C should be high enough to denature the PCR products and allelic ladder because each solution contained 15% formamide. However, when the cool DNA samples passed the cross junction at a speed of 50 µL/channel per min, denaturation was not sufficient due to slow (26) Zhang, N.; Yeung, E. S. J. Chromtogr., A 1997, 768, 135-141.

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Figure 5. Electropherograms showing simultaneous genotyping in eight channels from blood to final results. Injection: temperature 90 °C, voltage 150 V/cm, time 33 s. Separation: voltage 150 V/cm, effective length 60 cm, matrix 1.5% Mn 8 000 000 and 1.45% Mn 600 000 PEO solution. Laser: 15-mW 488-nm argon ion laser. Detection: CCD through a 520-nm long-pass filter, exposure time 300 ms. Capillaries in the array are labeled from 1 to 8 according to proximity to the excitation laser. Two blood samples were analyzed. Channels 1-4 show sample 1 and channels 5-8 show sample 2. The lower electropherogram is the vTT allelic ladder by itself. The genotype (asterisks) was identified by an increase in the relative intensities of the peaks within each locus when the sample is present.

heat transfer. Partial renaturing was observed at a junction temperature of 80 °C. A higher temperature such as 95 °C may be even better for denaturation. Temperatures higher than 90 °C were however not tested because of concerns of bubble formation from boiling and decomposition of the gel matrix (e.g., urea) at the capillary tips. The capillary reactors must be cleaned carefully after PCR and allelic ladder loading. An extremely small amount of the allelic ladder or the PCR products can be amplified easily by PCR during the subsequent run to cause interference. The washing step described in the Experimental Section was found to be highly satisfactory. This was demonstrated by performing negativecontrol PCR, in which water was used instead of blood while all other reagents were kept identical. No DNA fragments were found except for the primer peaks. The ability to reuse the array of reaction capillaries is vital to the ruggedness of an on-line system. After all, it is not just how fast or how many samples one can handle in one run, but also whether the operation can be repeated over and over again in rapid succession that is important for largescale applications. Multiplexed On-Line Genotyping. Figure 5 shows the results of automatic genotyping from blood to readable results in the multiplexed format. The peaks before 40 min are due to residual primers and fluorescent contaminants in the samples. As seen

from the electropherograms, purification before analysis was not necessary because the allele fragments are all above 100 bp. Channels 1-4 are results for blood sample 1, and channels 5-8 are results for blood sample 2. The electropherogram at the bottom is for the allelic ladder alone for comparison. The peaks were named by the number of the repeated sequence in each fragment. The vWA ladder has alleles from 13 to 20, THO1 has alleles from 5 to 11, and TPOX has alleles from 6 to 13. The genotype of blood sample 1 is clearly identified as (14, 17) in vWA, (8, 9) in THO1, and (9, 9) in TPOX. Blood sample 2 has the genotype (15, 17) in vWA, (9, 9.3) in THO1, and (8, 9) in TPOX. The 9.3 allele has one-base deletion from allele 10 in the THO1 locus.26 These two alleles were partially separated as shown in channels 5-8. The requirement of a single-base resolution factor of at least 0.5 at this size range makes this type of STR genotyping much more challenging than other PCR applications.4,27,28 The separation here can probably be improved by further optimizing the composition of the PEO polymer solution and/or by lowering the separation voltage, but such an effort was not necessary for allele identification. Direct PCR from blood often shows small contamination peaks especially in the vWA region. However, the true allele peaks were much more intense so that highly confident allele identification was still possible. The baseline fluctuations were more severe in channels 3 and 8. This may be related to unevenness in manual gel-filling and chemical noise in this run. The peak intensities of electropherograms also varied among the different channels even for identical blood samples. This is likely due to the variability of PCR efficiency, stacking injection efficiency, and nonuniform detection characteristics across the channels. However, such variations will not affect the identification of genotype as long as the PCR, injection, and detection provide enough signal to alter the relative peak intensities in each allelic region in our absolute standard method.13,15 In this sense, the PCR specificity is more important than its efficiency. Sometimes, contamination peaks will affect the assignment, such as a false result in the TPOX region in channel 7. For higher reliability, simple software can be used to automatically pick up the peaks and identify alleles based on the Q-test, as has already been demonstrated by Gao et al.15 in a 96-capillary array. CONCLUSIONS To the best of our knowledge, triplex PCR directly from blood in an array of fused-silica capillary microreactors was demonstrated for the first time. Multiplexed sample loading, reaction, standard addition, injection, separation, and detection were totally (27) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186. (28) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162.

integrated and automated. The present integrated system shows advantages of mutual compatibility in each step, speed, throughput, simplicity, and flexibility over current procedures. Simultaneous genotyping from blood to final readable results was achieved in eight parallel channels. The total time for one run is 2.5 h. However, since cleaning and regeneration of the separation capillaries can be implemented during the PCR reaction and since a subsequent set of PCR reactions can be run during electrophoretic separation of the first set of samples, the real cycle time can be shortened to 1.3 h. We note that, because of the concept of absolute standards,13,15 these results are obtained for one-color detection of the DNA fragments. Reoptimization of the PCR conditions should allow two or more sets (different loci) of multiplex PCR to be performed simultaneously by using two or more sets of differently labeled primers and multicolor detection. If so, forensic DNA fingerprinting at a crime scene can be achieved in 2.5 h with identification at error rates less than 1 in 108, which is the current legal definition of a unique match. Full integration and automation of direct PCR from blood was achieved here in capillary tubes. One can envision transferring this protocol to microfabricated devices4,27,28 or other solid-phase microreactors.29 Capillaries are more compatible with pressure flow for gel filling, for avoiding irreproducibility inherent to electroosmotic flow, and for sample purification in chromatographic columns.12 Capillaries are also more flexible during the research and development stage because minor modifications in the hardware do not require completely rebuilding the system. On the other hand, microfabricated devices do not require liquid valves (substituting instead multiple high-voltage relays), can be made more compact, and are more adaptable to mass production to reduce cost once the optimal protocol is established. Our future efforts will focus on incorporating larger numbers of channels and miniaturization of the reactor volume by reducing the capillary diameter to further lower the cost of reagents. Applications can be expanded using this concept to other sample preparation protocols prior to CE, such as drug screening and peptide mapping. 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. Received for review December 16, 1998.

October

19,

1998.

Accepted

AC981139J (29) Soper, S. A.; Williams, D. C.; Xu, Y.; Lassiter, S. J.; Zhang, Y.; Ford, S. M.; Bruch, R. C. Anal. Chem. 1998, 70, 4036-4043.

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