Multiple Sample PCR Amplification and Electrophoretic Analysis on a

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Anal. Chem. 1998, 70, 5172-5176

Multiple Sample PCR Amplification and Electrophoretic Analysis on a Microchip Larry C. Waters, Stephen C. Jacobson, Natalia Kroutchinina, Julia Khandurina, Robert S. Foote, and J. Michael Ramsey*

Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142

Polymerase chain reactions (PCRs) were carried out on as many as four DNA samples at a time on a microchip device. The PCR products were then analyzed, either individually or together on the same device, by microchip gel electrophoresis. A standard PCR protocol was used to amplify 199- and 500-base pair (bp) regions of bacteriophage λ DNA and 346- and 410- bp regions of E. coli genomic and plasmid DNAs, respectively. Thermal lysis of the bacteria was integrated into the PCR cycle. A product sizing medium, poly(dimethylacrylamide), and an intercalating dye for fluorescence detection were used in the electrophoretic analysis of the products. PCR product sizes were determined by coelectrophoresis with marker DNA. The ability to perform all of the steps of a biological assay on a single microchip promises significant advantages to biochemists and molecular biologists in terms of speed, cost, and automation.1 The development of devices that can perform multiplex polymerase chain reaction (PCR)2 and electrophoretic sizing is particularly interesting because of its potentially widespread application in molecular biology, clinical, and forensics laboratories3 for high-throughput genetic analysis. The first monolithic devices that integrated chemical reactions with analysis included microfluidic structures to mix reagents in nanoliter and subnanoliter reactors either prior to4 or after5,6 capillary electrophoresis. DNA restriction digestions with fragment sizing have also been performed on an integrated monolithic microchip.7 In a previous collaborative study, the use of microchip devices in the medical diagnosis of muscular dystrophy was demonstrated. This involved the amplification8 and microchip gel electrophoresis (MGE) analysis9 of the PCR products on separate microchip devices. A (1) Cheng, J.; Fortina, P.; Surrey, S.; Kricka, L. J.; Wilding, P. Mol. Diagn. 1996, 1, 183. (2) Chamberlain, S.; Gibbs, R. A.; Rainier, J. E.; Nguyen, P. N.; Caskey, C. T. Nucleic Acids Res. 1988, 16, 11141. (3) Erlich, H. A., Ed. PCR Technology. Principles and Applications for DNA Amplification; W. H. Freeman and Co.: New York, 1992. (4) Jacobson, S. C.; Hergenro¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (5) Jacobson, S. C.; Koutny, L. B.; Hergenro ¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472. (6) Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 4285. (7) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720. (8) Wilding, P.; Kricka, L. J.; Cheng, J.; Hvichia, G.; Shoffner, M. A.; Fortina, P. Anal. Biochem. 1998, 257, 95. (9) Cheng, J.; Waters, L. C.; Fortina, P.; Hvichia, G.; Jacobson, S. C.; Ramsey, J. M.; Kricka, L. J.; Wilding, P. Anal. Biochem. 1998, 257, 101.

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hybridized device that carries out DNA amplification via PCR in a silicon heater containing a polypropylene tube and electrophoretic analysis in a glass microchip has also been reported.10 Recently, the integration of DNA amplification and PCR product analysis on a single monolithic microchip device was demonstrated.11 The integrated steps included thermal lysis of bacteria to release DNA, simultaneous amplification of multiple gene loci using selected primer sets, and electrophoretic analysis of the amplified products. Previous studies have shown that many electrophoretic runs can be made in the same separation channel with a single filling of sieving medium.11 In addition, the analysis time is very short compared to the time that is currently required for sample DNA amplification. The most obvious way to take advantage of these features and to increase throughput of genetic analysis is to increase the number of DNA samples to be amplified on a single microchip. This report describes a microchip design and a procedure by which the complete analysis of multiple DNA samples, via PCR amplification followed by MGE analysis, can be made on a single microchip. EXPERIMENTAL SECTION Microchip Fabrication. The microchips were fabricated using standard micromachining techniques as previously described.5 The microchannel designs in Figure 1 were transferred onto the substrates using a positive photoresist, photomask, and UV exposure. The channels were etched into the substrate in a dilute, stirred HF/NH4F bath. To form the closed network of channels, a cover plate was bonded to the substrate over the etched channels by hydrolyzing the surfaces, bringing them into contact with each other, and processing thermally to 500 °C. Channel dimensions were measured using a stylus-based surface profiler prior to cover plate bonding, and the channel widths are reported at half-depth (see Figures 2- 5). The glass reservoirs (15- or 70-µL capacity) were affixed with epoxy at the point where the channel extends beyond the cover plate. The electroosmotic mobility was minimized by covalent immobilization of linear poly(dimethylacrylamide) (PDMA) on the channel walls,12 and the channels were filled with 4% (v/v) linear PDMA in 0.5× TBE (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA at pH 8.3). The sieving matrix was polymerized in the channels using 0.05% (10) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081. (11) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158. (12) Hjerten, S. J. Chromatogr. 1985, 347, 191. 10.1021/ac980447e CCC: $15.00

© 1998 American Chemical Society Published on Web 11/06/1998

Figure 1. (a) Schematic of microchip used for PCR amplification and electrophoretic analysis of multiple DNA samples. (b) Schematic of microchip used for sizing PCR products with a DNA marker.

ammonium persulfate and 0.2% TEMED. Microchips filled in this manner can be wrapped in polyethylene film and stored at 4 °C for at least 1 week prior to use. This sieving medium is replaceable, and multiple runs can be made with a single filling with good performance. This shows that the channel coating is stable and robust and that the separation polymer can be reproducibly generated by on-chip polymerization. Electrical contact to the reservoirs is made using platinum wire. PCR Amplification. The PCR reaction mixture contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 250 µg/mL bovine serum albumin, 200 µM each deoxynucleotide triphosphate, 1.0 µM each primer; 25 units/mL AmpliTaq DNA polymerase (Perkin-Elmer), and either λ DNA (10 ng/mL) or whole Escherichia coli cells. The E. coli strain was INV1aF′ (Invitrogen) containing plasmid pcDNAII. The target DNA in the plasmid was a cDNA clone of the plant gene agNt84.13 When whole E. coli cells were used, approximately two-thirds of a bacterial colony was collected with a sterile toothpick and resuspended in 50 µL of sterile H2O. This suspension was diluted 1:10 in the PCR reaction mixture. Approximately 12-µL aliquots of the reaction mixtures were placed in the PCR reservoirs of the PCR microchip (Figure 1a) for cycling. The 12-µL volume was used for convenience so that aliquots could also be analyzed by agarose slab gel electrophoresis for reference. All channels on the microchip and the other reservoirs contained sieving matrix in 0.5× TBE, and all reservoirs were topped with mineral oil to prevent evaporation. Amplification was carried out by thermally cycling the entire microchip in a commercial thermal cycler equipped with a slide griddle and a hot bonnet (MJ Research, Inc.). For DNA amplification, the temperature program was initiated at 94 °C for 4 min (to induce cell lysis where appropriate), and the subsequent temperature steps were 94 °C for 2 min, 50 °C for 3 min, and 72 °C for 4 min. These steps were repeated 24 times and then the temperature was held at 72 °C for 7 min to complete chain extension. Samples were cooled to 5 °C to conclude the program. Because sufficient amounts of PCR (13) Pawlowski, K.; Twigg, P.; Dobritsa, S.; Guan, C.; Mullin, B. C. Mol. PlantMicrobe Interact. 1997, 10, 656.

products were obtained from all of the DNA targets using the amplification program described, no attempt was made to further optimize these parameters. The primer set used to amplify a 199-bp segment of λ DNA was 5′-GGATACGTCTGAACTGGTCACG-3′ and 5′-GGCGCTGTGGCTGATTTCGATAACC-3′ (Life Technologies) and for a 500-bp segment of λ DNA was 5′-GATGAGTTCGTGTCCGTACAACTGG3′ and 5′-GGTTATCGAAATCAGCCACAGCGCC-3′ (Perkin-Elmer GeneAmp PCR Reagent Kit). The primer set used to amplify a 346-bp segment of the E. coli lamB gene was 5′-CTGATCGAATGGCTGCCAGGCTCC-3′ and 5′-CAACCAGACGATAGTTATCACGCA-3′ and for the 410-bp plasmid DNA target was 5′-ATGGGTTACTCCAAGACTTTTCTTCT-3′ and 5′-ATTAAGCTTGTATGCCAATAAAC-3′ (Life Technologies). Product Characterization. Following amplification an intercalating dye, TO-PRO (Molecular Probes, Inc.; is a potential mutagen), was added to the PCR reservoirs to a final concentration of ∼7 µM. The products were then loaded electrophoretically into the injection valve using the pinched sample injection technique.14 Simultaneous loading of multiple samples was effected by applying equal potentials to the appropriate reservoirs. Once they were loaded into the valve, the potentials were reconfigured for the separation.14 The microchip separations were monitored 2.5 or 3 cm downstream using a single-point detection scheme.7 The separation lengths and field strengths are indicated in each figure caption. An argon ion laser (5 mW, 514.5 nm) is focused to a spot on the separation channel using a lens (100 mm focal length). The fluorescence signal is collected using a 20× objective lens (NA ) 0.42), followed by spatial filtering (1.0-mmdiameter pinhole) and spectral filtering (514-nm notch filter with a 10-nm bandwidth, 540-nm band-pass filter with a 30-nm bandwidth), and measured using a photomultiplier tube. The data acquisition and voltage switching apparatus (interlocked for safety) used to accomplish these sizing measurements are computer controlled. After preliminary characterization of the PCR products by MGE on the PCR microchip (Figure 1a), they were sized by coelectrophoresis with a DNA marker on the PCR sizing microchip (Figure 1b). The DNA sizing ladder used in this study was a “50-bp” PCR marker preparation (12 mg/mL) containing six fragments of 50, 150, 300, 500, 750, and 1000 bp, each present at approximately equal weight/volume concentrations (Promega). From an earlier comparison of the electrophoretic mobilities of the fragments versus fragment size of this marker with those of the ΦX174 HaeIII digest DNA marker (Sigma), it was determined that none of the 50-bp marker fragments had substantial conformational variations which would make this marker unsuitable for this purpose.11 RESULTS AND DISCUSSION Using a microchip with a single PCR reservoir, it has been shown that multiplex PCR reactions can be carried out on a DNA sample, and the products analyzed by MGE on the same microchip.11 The ability to analyze a large number of DNA samples in this format would greatly enhance the utility of the technique. The analysis of multiple DNA samples in this format (14) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; J. M. Ramsey, J. M. Anal. Chem. 1994, 66, 1107.

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Figure 2. Electropherograms of the PCR products made from three DNA samples and analyzed on the same microchip. Targets: (a) λ DNA 500 bp; (b) plasmid DNA 410 bp; (c) genomic DNA 346 bp; (d) combined analysis of (a-c). The microchip (Figure 1a) was filled with 4% (v/v) PDMA, the separation length was 3.0 cm, and the separation field strength of 130 V/cm. The microchip channels were approximately 50 µm wide and 10 µm deep. The numbers denote fragment size in base pairs.

Figure 3. Electropherograms of the PCR products made from four DNA samples and analyzed on the same microchip. Targets: (a) λ DNA 199 bp; (b) genomic DNA 346 bp; (c) plasmid DNA 410 bp; (d) multiplexed genomic DNA 346 bp and plasmid DNA 410 bp. The microchip (Figure 1a) was filled with 4% (v/v) PDMA, the separation length was 3.0 cm, and the separation field strength of 130 V/cm. The microchip channels were approximately 50 µm wide and 10 µm deep. The numbers denote fragment size in base pairs.

is shown in Figure 2. Three DNA targets including λ phage (500bp) DNA and plasmid (410-bp) and genomic (346-bp) DNAs from whole E. coli cells were amplified in separate PCR reservoirs on the same microchip (Figure 1a). The PCR products were individually analyzed by sequentially applying voltage to each of the PCR reservoirs. No potential was applied to the reservoirs not being sampled. Products can be seen as single peaks with migration times consistent with their expected sizes (Figure 2ac). The three products can also be analyzed at once by simultaneous application of voltage to all three PCR reservoirs. The sample channels are designed to be of equal length, and consequently equal resistance, so that a single potential can be applied to run one or more samples. The expected three peaks can be seen in Figure 2d. This result illustrates how DNA targets from a variety of sources may be independently amplified in different reservoirs and then analyzed in a single electrophoretic run, provided the PCR product sizes are appropriately spaced for the resolution by MGE. [The fourth PCR reservoir in this experiment contained the multiplexed 346- and 410-bp targets. Because the 346-bp target was not significantly amplified in this reservoir, it became a duplicate of another sample (Figure 2b) and is not shown again.] In another experiment, four DNA samples were analyzed on a single microchip. DNA targets were λ phage (199-bp) DNA and genomic (346-bp) and plasmid (410-bp) DNAs from whole E. coli cells, with the latter two also multiplexed in a separate reservoir. MGE analysis of these PCR products is shown in Figure 3. Again, the products can be seen as single peaks, or double in the case of the multiplexed reaction, with migration times consistent with their expected sizes. Periodically, minute

amounts of the products present in the unsampled reservoirs were observed in the electropherograms, e.g., the 199- and 346-bp products as seen in Figure 3c. This is due to slight leakage from the reservoirs not being sampled and can be prevented by actively controlling the potentials applied at those reservoirs. Although the migration times of the PCR products can be quite reproducible, as is indicated for the 346- and 410-bp products shown in Figure 3b-d, a more accurate way to estimate the size of the PCR products is by coelectrophoresing the products with a known DNA marker. The microchip whose design is illustrated in Figure 1b can be used to analyze the sizing marker, the amplified product, or a mixture of the two. The contents of the marker and PCR products reservoirs (Figure 1b) can be proportioned in any desired ratio simply by applying the appropriate voltages. In Figure 4, this is tested by analyzing the sizing marker (50, 150, 300, 500, 750, and 1000 bp) using 4% PDMA sieving medium, with no potential applied to the PCR product reservoir (Figure 4a). Then, the PCR products and the marker are proportioned equally at the mixing tee for injection onto the separation column (Figure 4b-e). As expected, the 199-bp product migrates between the 150- and 300bp markers, at 144 s (Figure 4b). The 346- and 410- bp products migrate at 131 and 138 s, respectively, which is between the 300and 500-bp markers and is consistent with their expected sizes (Figure 4c,d). These migration times are reproduced in the case of the multiplexed 346- and 410-bp products (Figure 4e). From individual plots of electrophoretic mobilities of the fragments versus fragment lengths for the data shown in Figure 4, the experimentally determined sizes of the PCR products were [expected/measured (% deviation)]: 199/187 (-6.0%), 346/331

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Figure 5. (a) Electrophoretic mobilities of the PCR products in Figure 2 (b) and the 50-bp sizing ladder (O) vs fragment size. These data (not shown) were generated on a microchip (Figure 1b) with channel dimensions of 50 µm wide and 9 µm deep. (b) Electrophoretic mobilities of the PCR products in Figure 3 (9) and the 50-bp sizing ladder (0) versus fragment size. The data are extracted from the electropherograms shown in Figure 4. The lines represent a smooth fit to the data from the respective microchips.

(-4.3%), and 410/400 (-2.4%) (Figure 5a). The PCR products shown in Figure 2 were similarly sized (data not shown) with the following values determined: 346/333 (-3.8%), 410/400 (-2.4%), and 500/507 (+1.4%) (Figure 5b). Run-to-run reproducibility on the sizing microchip was very good. The percent relative standard deviation of the migration times for eight runs of the eight DNA fragments in the sample depicted in Figure 4e was better than 0.3% in all cases. The preceding data show that PCR product sizing, using the microchip format illustrated in Figure 1b, is very reproducible. The accuracy of sizing the 500-bp product is greater than 98% as was noted previously.11 A number of factors might explain the relatively poorer concordance of the expected versus measured values determined for the smaller products. It is unlikely that the 50-bp DNA marker exhibits anomalous electrophoretic mobilities at those sizes less than ∼500 bp because the mobility versus size plot of this marker fits well with that obtained with the ΦX174 HaeIII digest DNA marker.11 It is known, however, that some DNA molecules may contain sequence variations which alter their conformation and, consequently, their electrophoretic mobilities.15,16 For example, the 587-bp HaeIII fragment from pBR322 (Sigma) consistently migrates slower than the 603-bp HaeIII fragment from ΦX174 in this system. Other factors such as

separation matrix, temperature, buffer strength, column coating, and separation field strength can alter the electrophoretic mobility of DNA. However, because a format of coelectrophoresis is used, these factors are effectively neutralized. In any case, with this type of DNA analysis, it is most often not the absolute size of the product made but the relative sizes of PCR products made from different DNA samples that are important. In most cases, such differences would be revealed by coelectrophoresis of the samples. The ability to amplify and analyze multiple DNA samples on a single microchip has been demonstrated. By keeping the fluidic structures simple in design and fabrication, these devices eventually could be disposable units which would then eliminate problems associated with reuse, such as DNA carry-over. The potential also exists for even higher throughput. For example, the peak capacity of the separations shown here would easily allow the separation of at least 10 PCR products between the sizes of 50 and 500 base pairs. Thus, in a high-throughput application of PCR analysis, primers could be designed so that 10 or more PCR products could be analyzed simultaneously. The multiple targets could be amplified in a single reservoir or, in situations where primer or other reaction condition incompatibilities exist, in individual reservoirs. In either case, all of the PCR products can be analyzed in a single MGE run by injecting from one reservoir or from multiple reservoirs using an appropriate microfluidic design. Still higher throughput can be achieved by further increasing the peak capacity of the electrophoretic analysis and the sample well count. The latter was demonstrated recently, whereby the products from 96 PCRs were electrophoretically analyzed on a single 10-cm microchip device.17 Numerous applications for microchips of this design can be envisioned. Those involving PCR-based analyses of markers used

(15) Berka, J.; Pariat, Y. F.; Muller, O.; Hebenbrock, K.; Heiger, D. N.; Foret, F.; Karger, B. L. Electrophoresis 1995, 16, 377. (16) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990, 516, 33.

(17) Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2256.

Figure 4. Electropherograms of the PCR products (filled peaks), amplified and analyzed on a single microchip and shown in Figure 3, mixed with a 50-bp DNA sizing ladder (unfilled peaks). The microchip (Figure 1b) had channels that were approximately 74 µm wide and 10 µm deep. It was filled with 4% (v/v) PDMA, the separation length was 2.5 cm, and the separation field strength was 120 V/cm. The numbers denote fragment size in base pairs.

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in genetic mapping18 and forensic analysis19 are particularly appropriate. Clinical diagnoses of inherited diseases, for which the involved genes have been identified, and detection of infectious microorganisms are other applications for which these microchips can be used. One constraint to performing multiple PCRs on the same microchip, as described in this paper, is that all DNA targets must amplify under the same cycling conditions. This will not be a problem for the applications mentioned above because, while the DNA samples will vary, the primers and the DNA targets remain the same. In these applications, the microchip offers a rapid and potentially inexpensive means of manipulating and analyzing DNA in a way that minimizes sample handling and the potential for contamination.

ACKNOWLEDGMENT This research is sponsored by ORNL Laboratory Directed Research and Development. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC05-96OR22464. J. K. and N.K. were supported through appointments to the ORNL Postdoctoral Research Associates Program, administered by ORISE and ORNL. The authors thank Justin E. Daler and Judith Eggers for assistance in fabricating the microchips. We also thank Dr. Beth C. Mullin (The University of Tennessee at Knoxville) for providing the bacteria used in this work.

Received for review April 24, 1998. Accepted September 24, 1998. (18) Taylor, B. A.; Navin, A.; Phillips, S. J. Genomics 1994, 21, 626. (19) Jeffreys, A. J.; Wilson, V.; Thein, S. L. Nature 1985, 314, 67.

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