42 Micro¯uidic devices for DNA sequencing: sample preparation and electrophoretic analysis Brian M Paegel, Robert G Blazej and Richard A Mathiesy Modern DNA sequencing `factories' have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inef®cient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a `sequencing lab-on-a-chip'. Integrated micro¯uidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template ampli®cation, Sanger extension, puri®cation, and electrophoretic analysis in a single micro¯uidic circuit is now attainable. Addresses Department of Chemistry and UCB/UCSF Joint Bioengineering Graduate Group, University of California, Berkeley, CA 94720, USA y e-mail: [email protected] Current Opinion in Biotechnology 2003, 14:42±50 This review comes from a themed issue on Analytical biotechnology Edited by Norm Dovichi and Dan Pinkel 0958-1669/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0958-1669(02)00004-6 Abbreviations BAC bacterial arti®cial chromosome CAE capillary array electrophoresis lCAE microfabricated capillary array electrophoresis JGI Joint Genome Institute LPA linear polyacrylamide PCR polymerase chain reaction PRS polymorphism ratio sequencing RTD resistive temperature detector Introduction The initial goals of the Human Genome Project are rapidly approaching completion [1±3]. While this project is just ®nishing, the completion of a myriad of other model organisms [4] and the understanding of sequence-dependent organismal variation [5] will be more feasible once methods for decreasing cost, analysis time, and reliance on cumbersome and expensive robotics have been developed. In addition to increasing sequencing capacity, this new objective will also liberate the technology to address more diverse comparative sequencing and polymorphism discovery and screening needs. Current Opinion in Biotechnology 2003, 14:42±50 However, the routine use of electrophoretic sequencing in these higher-throughput arenas is currently impossible because of the cost of the macro-process, estimated to be $0.10±0.50 per reaction depending on scale. The Joint Genome Institute (JGI), which is representative of streamlined modern production sequencing facilities, has developed an automated process for preparing, purifying, and analysing DNA sequencing samples. Figure 1 presents their sequencing chemistry ¯ow chart, illustrating the volume and time scale involved at each step. The chart starts with the transformation step; electro-competent cells are transformed with a pUC19 sequencing vector containing a 1 kbp insert from a large-clone library, such as a bacterial arti®cial chromosome (BAC). The transformed cells are plated and grown for 18 h before automated colony picking. Inserts are ampli®ed isothermally in a 10 ml reaction using rolling circle ampli®cation [6], sequenced in a 10 ml reaction using Sanger dideoxy dye-terminator chemistry [7], puri®ed from template and free dye-terminators on magnetic carboxyl beads [8], and analysed on a MegaBACE capillary array electrophoresis (CAE) sequencer. This robotically driven aÁ la carte sequencing process is problematic in terms of space and maintenance, and sample transfer reliability. Furthermore, the enzymemediated reactions, including Sanger sequencing and template ampli®cation, consume costly reagents at high volume to meet liquid handling limitations. If these limitations were to be removed, how far could we scale down the volumes in the Sanger sequencing process in principle? Starting with a single transformed Escherichia coli cell containing ®ve copies of the sequencing vector (a conservative assumption), 30 cycles of PCR result in 109-fold ampli®cation, generating 10 fmol of template; a 500 nl Sanger sequencing reaction would require only 1 fmol of ampli®ed plasmid DNA. Assuming a minimum yield of 50% for 30 sequencing thermal cycles, 150 fmol of product is generated. When concentrated to 10 mM (corresponding to 10 nM per peak for a sequencing ladder of 1000 bands), this amount of ready-to-inject product occupies 15 nl. The average plug volume injected on a capillary is only 1 nl. We therefore conclude that sequencing a vector contained in a single transformed cell is feasible. Can this ambitious goal be achieved in practice? The nanoliter volumes intrinsic to such a micro-sequencing process are inaccessible to current CAE-based sequencing platforms (Figure 1). Nanoliters are, however, www.current-opinion.com Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 43 Figure 1 10 cm Transformation 25 µL 2h Plate and grow cells 300 mL 20 h 2m Pick colonies 10 mL 10 min 30 cm DNA amplification 10 µL 18 h Sanger extension 10 µL 120 min 40 cm Product purification 30 µL 60 min 1m Electrophoresis 15 µL 150 min Current Opinion in Biotechnology A flowchart for conventional macroscale DNA sequencing sample processing and electrophoretic analysis. Instrumentation, including scale bars to indicate approximate instrument size, is shown on the left www.current-opinion.com the ideal volume for a microchip sequencer that performs on-chip enzymatic reactions, sample transport, and electrophoretic analysis. Here, we review recent demonstrations of microchip sample analysis and processing in the context of enabling this micro¯uidic `sequencing lab-ona-chip'. Microfabricated electrophoretic DNA sequencing separation devices In 1995, Woolley and Mathies [9] demonstrated that narrow sample zone injection on a microdevice greatly reduces the column length required to separate DNA sequencing fragments; 200 bases were called from a fourcolour sequencing sample using a 3 cm long microchannel in only 9 min. Presently, work on single-channel microchip electrophoresis applied to DNA sequencing has focused on improving data quality and read length by incorporating well-characterized, high-molecular weight linear polyacrylamide (LPA) sieving matrices, ef®cient sequencing chemistries utilizing energy-transfer (ET) ¯uorescent reagents, and microchannels with increased separation length. By extending the channel separation length from conventional microchip values of 3 to 7 cm and synthesizing sequencing fragments with improved ET reagents, 500 bases (99% accuracy) were acquired in only 20 min [10]. Similar experimental conditions applied to an 11.5 cm long channel produced an average of 505 bases in 27 min from human chromosome 17 samples [11]. Further improvement of single-channel separation devices was accomplished by implementing a blended, emulsion-polymerized LPA sieving matrix [12] on long, straight channels. The basic channel schematic and results for these devices are summarized in Figure 2. A standard cross-injector with an offset of 150 mm is used to increase the amount of sample injected (Figure 2a). An 11.5 cm long version of this device produced 580 bases (98.5% accuracy) in 18 min [13]. For longer read lengths, the channel length can be increased to 40 cm to acquire 800 bases, although the run time is substantially increased to 80 min [14]. Plots of resolution versus base position for both devices are presented in Figures 2b and 2c. Bases with a resolution >0.5 were considered to be of high data quality, although phred (base-calling and data accuracy evaluation software) analysis is now used exclusively to describe sequencing data quality [15,16]. An even more attractive advantage of the microfabricated platform is the ease with which intricate, ultra-dense channel arrays can be constructed. Although conventional CAE-based sequencers are substantially faster than slab-gel-based instrumentation, their predominant contributions to genomic sequencing lie in their capacity to provide a higher degree of automation and analysis of each process step. Space measured in many square metres is required to accommodate the supporting instrumentation. (Protocols taken from http://www.jgi.doe.gov/Internal/prots_index.html.) Current Opinion in Biotechnology 2003, 14:42±50 44 Analytical biotechnology Figure 2 Sample (a) Cathode In 2000, a prototype microfabricated capillary array electrophoresis (mCAE) DNA sequencing device consisting of an array of sixteen 7 cm long channels was presented [19]. The 16 cross-channels were fanned out on a 100 mm diameter wafer, each channel having its own cathode, sample, and waste wells. All channels converge on a common high-voltage anode. An average of 457 bases per lane (99% accuracy) was acquired in 15 min. Other array structures for DNA sequencing were also presented around this time, but lacked the cross-injector, a requirement for high-performance, high-speed microchip DNA sequencing [20,21]. Waste (b) 1.2 Leff = 11.5 cm 18 min Resolution 1.0 0.8 0.6 0.4 0.2 100 300 500 700 Base number (c) 1.2 Leff = 40 cm 78 min Resolution 1.0 0.8 0.6 0.4 0.2 0 200 400 600 Base number 700 1000 Straight-channel DNA sequencing chip design and resolution results. (a) The standard cross-injector sample and waste channel arms are offset to increase the injected plug length. (b,c) Plots of sequencing fragment resolution versus base position show device performance; a resolution of >0.5 is considered high quality. Increasing the device length from (b) 11.5 cm to (c) 40 cm increases DNA sequencing read length at the expense of increased analysis time. Reproduced with permission from [13] and [14]. Current Opinion in Biotechnology 2003, 14:42±50 multiplexing. Microfabrication removes limitations associated with assembling multi-capillary arrays, allowing micron-precision, scalable control of channel layout and arrangements [17,18] in addition to the integrated reactor structures to be discussed later. A 96-lane mCAE DNA sequencing bioprocessor was introduced in 2002 [22], the ®rst massively parallel micro¯uidic circuit design (Figure 3) exploiting microchip injectors for high-performance separation. The chip is radially symmetric, composed of 48 doublets containing unique sample wells and common cathode and waste wells [23]. The serpentine channel leading toward the common, high-voltage anode at the centre of the device contains four hyperturns and provides the necessary separation length for performing DNA sequencing in LPA. The hyperturns minimize geometric band dispersion introduced by turns in the separation path [24±26]. The separation length is extended from 5.5 cm for a straight channel on a 150 mm diameter wafer to 15.9 cm. Filling long channels with gel-sieving matrix required the use of a high-pressure loading and cleaning device [27], although novel copolymer matrices exhibiting thermoresponsive viscosity may alleviate this requirement [28]. Detection is accomplished using a rotary confocal ¯uorescence scanner that interrogates all lanes sequentially [23]. The 96-lane mCAE device acquired 41 000 bases (99% accuracy) in only 24 min [22], a sequencing throughput ®vefold higher than currently deployed systems. The same bioprocessor was subsequently used in the development and implementation of a novel, sequencing-based polymorphism identi®cation and screening assay called polymorphism ratio sequencing (PRS) [29]. In this study, the entire human mitochondrial genome was screened by PRS in a 30 min run of the sequencing bioprocessor that identi®ed 30 known and six previously unknown polymorphisms. This work demonstrated the versatility of the mCAE processor as well as the value of a comprehensive sequencing-based assay. Micro¯uidic DNA puri®cation Electrophoretic analysis of the DNA sequencing products, although challenging from the standpoint of separation science, is only the ®nal step in an involved series www.current-opinion.com Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 45 of chemical processing steps, as presented in Figure 1. Puri®cation of DNA sequencing products, the step just before electrophoretic analysis, is critical. Residual background ions (buffer, chloride, unincorporated dideoxynucleotides) present in the sequencing extension reaction cocktail after thermal cycling reduce injection ef®ciency, signi®cantly degrading sequence signal-to-noise [30]. Frequently, either ethanol precipitation or more rigorous spin-column methods are used to purify the extension products [31,32], although for high-throughput operations, both pose serious automation problems because centrifugation is required. The ideal microchip puri®cation circuit should be fully integrated, low volume, and, most importantly, eliminate manual sample transfer and centrifugation steps. Figure 3 (a) (b) Sample Cathode Waste (c) Anode The 96-lane DNA sequencing bioprocessor. (a) Forty-eight doublet structures (96 samples) are radially arrayed on a 150 mm glass wafer around a common, central anode. (b) The injector region of a doublet contains two unique sample wells, and common cathode and waste wells. Cross-injection is achieved by applying a positive potential to the waste well while grounding the sample well, which fills the 1 nl intersection with DNA. Switching to grounded cathode, small positive potentials on sample and waste wells, and high voltage at the anode draws a small plug of DNA down the serpentine channel for separation. (c) A tapered turn, or hyperturn, allows the incorporation of serpentine channel geometries without introducing geometric www.current-opinion.com Capillary-based systems containing serial separation steps provided initial indications of how puri®cation might be coupled to electrophoretic analysis of DNA sequencing products. In a study from Yeung's group [33], capillary zone electrophoresis was used to isolate extension fragments in free solution. The isolated band was then directly injected onto a manually coupled DNA sequencing capillary ®lled with gel sieving matrix. However, much like bundling capillaries to form arrays, joining capillaries is problematic because it requires individual intricate glass joints, and also this method requires complex timing for band extraction. Microfabricated systems offer more precise electrophoretic control of analyte, and band extraction channels can easily be fabricated to accomplish micro-preparative fraction collection, although band extraction timing is still challenging [34]. In other work toward the goal of chemistry integration, Tian et al. [35] developed a low-volume ¯ow cell containing a variety of silica resins for solid-phase extraction of DNA from cell lysate followed by off-chip PCR veri®cation. Their silica-resin-containing 500 nl bed had a recovery ef®ciency of 70% for DNA collected from white blood cells, removing >80% of contaminating proteins in 10 min. This work was later expanded to a microfabricated ¯ow cell containing sol-gel/bead composites with similar DNA extraction ef®ciencies and analysis times [36]. Another intriguing puri®cation method relying on analyte focusing was demonstrated in which the analyte electrophoretic migration is modulated spatially by establishing a temperature gradient along the path of travel. Since electrophoretic mobility is temperature dependent and bulk electroosmotic ¯ow is constant, a point in the microchannel exists such that the analyte electrophoretic velocity is exactly countered by the bulk ¯ow, resulting in analyte concentration. A variety of ¯uorescently tagged dispersion from the turn. The separation channel shown here contains four hyperturns with a total separation length of 16 cm. Reproduced with permission from [22]. Current Opinion in Biotechnology 2003, 14:42±50 46 Analytical biotechnology molecules, including DNA, were isolated and concentrated as high as 10 000-fold in this manner [37]. An alternative approach for on-chip puri®cation is hybridization-mediated capture, which is perhaps the most effective and stringent isolation method for nucleic acids. Capture probes immobilized in a gel-phase support can be used for size- and sequence-selective isolation of nucleic acids. Olsen and coworkers at NIST [38] have demonstrated the use of DNA hydrogel plugs polymerized inside a microchannel for selective capture and 100fold concentration of a 150 nM solution of TAMRAlabeled oligonucleotide complementary to the hydrogel in 25 min. A similar concept was recently utilized in our group [39] to develop the integrated microdevice for DNA sequencing shown in Figure 4. A 60 nl chamber is coupled to a microchip injector and serpentine sequencing channel. The chamber is ®lled with a replaceable acrylamide-methacryl-DNA capture copolymer. The gel-immobilized capture probe sequence is chosen such that it is complementary only to sequencing extension fragments, thus providing selective capture. Sample is electrophoretically driven through the chamber, preconcentrating and desalting only the extension products while passing excess non-complementary primer and ionic contaminants. The product is electrophoretically washed with fresh buffer, and then released by raising the temperature of the chip to the sequencing process temperature. Standard cross injection is followed by separation. The total chamber volume is more than 100-fold lower than macroscale puri®cation reactions and results in a 200-fold volumetric concentration of sample. Puri®cation of the sequencing products is accomplished in 120 s, a tenfold decrease in puri®cation time over conventional methods. Furthermore, this process requires no centrifugation, integrates sample transfer steps, and generates sequencing peaks that are 85% of the intensity of ethanol-precipitated sequencing product [39]. Submicroliter PCR and Sanger sequencing Several strategies for performing low-volume Sanger cycle sequencing in capillaries have been presented that build on the previously established feasibility of thermally cycling premixed Sanger sequencing reagents in sealed capillaries using fast, convection-based cyclers [40,41]. Hadd and coworkers [42] demonstrated that 500 nl dye-terminator reactions prepared in a highthroughput 96 capillary reactor array could be rapidly cycled, blown out of the capillary reactor, precipitated, and analysed on a commercial sequencer to yield results comparable to the macroscale sequencing process. Xue and coworkers [43] demonstrated even lower volume (100 nl) reactions inside a capillary. These studies establish the feasibility of nanoliter-scale sample preparation, but because of manual coupling, their integration is problematic. Current Opinion in Biotechnology 2003, 14:42±50 Figure 4 (a) Inject Bind Separate + + S + + (b) (c) (d) 5 10 15 20 Time (min) 25 30 Integrated nanoliter affinity extraction sample cleanup and preconcentration followed by electrophoretic analysis. (a) The sample purification circuit contains a 60 nl extraction chamber coupled to standard cross injection fluidics. Crude product (well `S') is electrophoretically driven through the chamber, which contains an affinity extraction acrylamide matrix. Extension products are selectively bound and washed with electrophoresis buffer. For cross injection, the sample is brought to the sequencing process temperature (T 678C > TM ), melting the matrix-product duplex, and injection proceeds as described in Figure 3. (b±d) The C terminations from four-colour sequencing runs illustrate the difference between sample purified by (b) on-chip affinity extraction, (c) off-chip ethanol-precipitated sample, and (d) directly injected crude product. The excess primer and first four terminations (highlighted in black) have no specificity for the capture matrix and are therefore absent from the on-chip-purified sample. Reproduced with permission from [39]. www.current-opinion.com Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 47 On-chip template ampli®cation has been presented in a variety of formats based primarily on PCR. Recent work has explored alternative methods for thermally cycling ultra-low volumes with expanded temperature control and increased thermal ramping rates. Liu and coworkers [44] showed that as little as 12 nl of PCR mix could be peristaltically pumped through three thermal zones on a polydimethylsiloxane (PDMS) device by activating onchip valves in a rotary fashion. PCR ampli®cation was successful, although the starting template concentration of 2 ng/ml was relatively high for a micro¯uidic reactor (approximately 3 million template copies in the reactor). Similarly, contactless heating using an infrared diode laser provided a means for cycling only 5 nl with heating times of >508C/s [45]. The ®rst monolithic, nanoliter-scale DNA ampli®cation system integrated with electrophoretic analysis was presented by Lagally et al. [46]. The system consists of a 300 nl reactor ®tted with a polyimide foil heater and thermocouple for reactor temperature control, and coupled directly to a high-speed microchip electrophoresis circuit to provide integrated analysis of the PCRampli®ed product. Twenty thermal cycles were performed in 10 min, and the product was injected, separated, and detected in 90 s. The reactor ampli®ed as few as 20 starting copies of template with an extrapolated limit of detection of two copies [46]. Subsequent elaboration of the device design included the incorporation of microfabricated thin-®lm heaters and platinum resistive temperature detector (RTD) temperature control elements. A schematic of this system is presented in Figure 5, where the chamber with microfabricated heating elements and RTD elements is shown in the magni®ed inset. The heater is fabricated on the outer surface of the device whereas the RTD is fabricated so that it is in direct contact with the ¯uid inside the nanoliter reactor, thus providing exquisite control over process temperature [47]. The device is capable of stochastic single-template ampli®cation [48], greatly exceeding the reaction sensitivity requirements for on-chip template preparation from a single transformed cell. Figure 5 (a) Heater leads (backside) RTD leads 4-Lead RTD S C W V Reactor Heating coils A (b) RTD leads Reactor Heater A thermal cycling reactor integrated with microchip electrophoresis circuitry. (a) The reactor (green) addressed on the right by microfabricated heating elements (red) and 4-lead RTD (blue) for sensing and controlling process temperature. The reactor volume is sealed by a valve at the top (S) and a hydrophobic vent at the bottom (V). The left side of the reactor is coupled to an injection cross with cathode (C), anode (A), and waste (W) wells. (b) The side view shows the layer structure of the device with the RTD (blue) inside the glass (hatched) reactor. The heater (red) is fabricated on the back of the chip. Adapted from [47]. Integrated cell sorting The front end of DNA sequencing, requiring lengthy culture growth steps and colony picking with expensive and macroscale robotics, represents the most time-inef®cient and complicated portion of the process. Sorting individual transformants directly from the electroporated culture to sequence ampli®cation reactors obviates these constraints, but will require precisely driven ¯uidics capable of sensing single transformed cells and must be compatible with culture media. Microfabricated cell sorters generally consist of a ¯uidic driving mechanism (electrokinetic or hydrodynamic) leading to a T- or Y-shaped intersection, which connects www.current-opinion.com waste and collection reservoirs. Electrokinetically driven ¯uidics sort by switching the driving voltage source between the target and waste arms [49]. Hydrodynamically driven ¯uidics (Figure 6) sort using valves [50] on each arm to determine the destination well. Pressuredriven systems are preferable for cell sorting because buffers used in electrokinetically driven systems may be incompatible with the culture, and the high ionic strength of culture media does not provide a favourable double-layer for establishing effective electroosmotic ¯ow. In addition to eliminating buffer compatibility issues, bi-directional peristaltic pumping can be used Current Opinion in Biotechnology 2003, 14:42±50 48 Analytical biotechnology Figure 6 Figure 7 1 cm Collection Waste V2 Fluorescence V1 Transformation 25 µL 2h Sort cells 5 µL 20 min V2 open V1 open Time Peristaltic pump 1 cm DNA amplification 300 nL 10 min PDMS valve Sanger extension 300 nL 20 min Input culture A PDMS cell sorting device with integrated valves. The culture to be sorted is introduced at the bottom of the structure. A channel leads to a Y intersection with output arms to collection and waste reservoirs. Pneumatically actuated PDMS valves V1 and V2 control flow through the arms. Cells are driven through the structure by peristaltic pumping accomplished by serially actuating a series of valves before the sorting intersection. The inset shows fluorescence signal and corresponding valve states for a fluorescence-activated cell sorting strategy. Adapted from [51]. to trap a cell in the detection volume for signal con®rmation, demonstrating the precision single-cell handling capabilities necessary for sorting individual sequencing transformants [51]. Integration of these all-PDMS devices with electrophoretic analysis may be problematic as PDMS is not well characterized for electrophoresis, DNA sample loss through absorption could be considerable, and polymer auto¯uorescence will signi®cantly increase detection limits. Recently, Grover and coworkers [52] presented new fabrication methods for reliably producing valves to control ¯uid ¯ow in all-glass CE channels using PDMS membrane multi-layer structures. The biggest challenges to the microsequencing paradigm proposed here lie in these initial cell-handling steps. The success of its evolution will hinge on the development of integrated and scalable sorting strategies. Conclusions We have dissected the modern DNA sequencing production line into its most basic and necessary elements. Each Current Opinion in Biotechnology 2003, 14:42±50 1 cm Product purification 60 nL 2 min 15 cm µCAE 10 nL 24 min Current Opinion in Biotechnology A microfabricated `sequencing factory-on-a-chip'. An individual subclone is sorted from a transformed culture and directed to a PCR amplification chamber. Single-cell amplification is followed by template purification. Purified template is moved to a thermal cycling reactor for generation of sequencing extension fragments. The extension fragments are isolated, preconcentrated, and injected into an electrophoretic analysis microchannel. The total estimated process volume from cell to called bases is 1 ml. www.current-opinion.com Microfluidic devices for DNA sequencing Paegel, Blazej and Mathies 49 part can be executed on much lower volume and time scales when integrated into a micro¯uidic device. We anticipate the development of an integrated processor that can take a single transformed cell directly to called bases using less than 1 ml of reagent in total. This microsequencing process, shown schematically in Figure 7, will exhibit entirely on-chip sample transport between steps. Nanoliter-scale enzymatic reactions that are completed in minutes and linked directly to product puri®cation, followed by high-speed microchip electrophoretic analysis will be all employed. Analysis times can be expected to drop an order of magnitude and reaction volumes can be expected to drop two or more orders of magnitude. The development of such a microfabricated `sequencing factory-on-a-chip' will bring high-throughput DNA sequencing to the everyday biology laboratory of tomorrow. Acknowledgements This work was supported by grants from the National Institutes of Health (HG01399) and from the Director, Of®ce of Science, Of®ce of Biological and Environmental Research of the US Department of Energy under contract DEFG91ER61125. BMP was supported by a NIH trainee fellowship from the Berkeley Programme in Genomics (T32 HG00047). References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Collins FS, Patrinos A, Jordan E, Chakravarti A, Gesteland R, Walters L, Fearon E, Hartwelt L, Langley CH, Mathies RA et al.: New goals for the US human genome project: 1998-2003. Science 1998, 282:682-689. 2. 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