DNA Analysis Using an Integrated Microchip for Multiplex PCR

Jul 28, 2014 - DNA Analysis Using an Integrated Microchip for Multiplex PCR ... Versailles − Saint Quentin en Yvelines University, 55 Avenue de Pari...
0 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/ac

DNA Analysis Using an Integrated Microchip for Multiplex PCR Amplification and Electrophoresis for Reference Samples Delphine Le Roux,† Brian E. Root,‡ Carmen R. Reedy,‡,¶ Jeffrey A. Hickey,‡ Orion N. Scott,‡ Joan M. Bienvenue,§,# James P. Landers,‡,∥,⊥ Luc Chassagne,† and Philippe de Mazancourt*,† †

Versailles − Saint Quentin en Yvelines University, 55 Avenue de Paris, 78000 Versailles, France MicroLab Diagnostics, Inc, 705D Dale Ave., Charlottesville, Virginia 22903, United States § Lockheed Martin Corporation, 6801 Rockledge Drive, Bethesda, Maryland 20817, United States ∥ Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22908, United States ⊥ Department of Mechanical Engineering, University of Virginia, Engineer’s Way, Charlottesville, Virginia 22904, United States ‡

S Supporting Information *

ABSTRACT: A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

O

the development of new strategies, including rapid approaches to the subassays (e.g., rapid PCR),2−5 robotic systems for high throughput, and integrated microfluidic systems. Micrototal analysis system (μTAS) technologies have been shown to be effective for both genetic6 and forensic DNA analysis.7 Microfluidics offers several advantages, including a cost-saving reduction in reagent volume, faster processing times of individual subassay steps, minimal operator intervention if the system is automated or semiautomated, and elimination of potential contamination as a result of the “closed” nature of integrated systems. A major challenge in creating sample-toanswer microfluidic systems has been the integration of all of the chemistries at microscale volumes in a single monolithic device where the physically isolated processes can be fluidically connected. One of the first examples of this capability was described by Easley et al. in which a glass microfluidic chip

ver the last two decades, the analytical community has been challenged to provide faster results and in an increasingly simpler format relative to the conventional methodologies currently in use in the forensic laboratories. The current forensic methods for human identification (HID) based on short tandem repeat (STR) analysis1 require multiple instruments and highly trained scientists to perform each of the requisite steps, DNA extraction, quantitation, multiplex PCR amplification, and capillary electrophoresis separation, needed to generate a CODIS-uploadable genetic profile. While STR analysis provides unparalleled discriminating power to uniquely identify each person, the analysis is currently performed at centralized forensic laboratories. This significantly increases the time to obtain results and eliminates the ability to make realtime decisions in critical, time-sensitive cases. The forensic community is in need of advances in technology that provide the genetic result faster and simplify the process so that the analysis can be performed at the point of sample collection. The time and cost associated with performing an STR analysis with current methodologies are major driving forces in © 2014 American Chemical Society

Received: April 18, 2014 Accepted: July 28, 2014 Published: July 28, 2014 8192

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

Therefore, this demonstrates a critical step toward inexpensive microchips for either partial or full integration of sample processing.

integrated on-chip DNA purification and PCR-based amplification, followed by separation and detection for an infectious pathogen.6 With HID genotyping utilizing the same basal processes, a comparable device would have obvious advantages. The multistep analysis, utilizing multiple instruments that currently requires hours or days, could be accomplished in an expedited manner in an automated, closed system devoid of opportunity for contamination and operated by minimally trained users. Microfluidic chips for the individual STR analysis steps have previously been demonstrated. Price et al.8 provided a review of nucleic acid extraction techniques on microchips such as the adaptation of solid phase extraction to different chip substrates.9−11 Similarly, various PCR approaches12,13 and electrophoretic separations14−16 have been demonstrated in microdevices. In the past decade, the trend has been to integrate all the steps required for genetic analysis. Integration of two of the processes used for a STR typing analysis has been described.17−21 However, one of the most significant challenges has been to achieve the stringent DNA separation resolution and precision requirements on a microfluidic chip. To achieve rapid, microchip STR separations, several groups have performed investigations using the commercial Agilent 2100.22−24 However, in each case, the resolution and precision of the separation was found to be insufficient for forensics metrics and accurate allele calling. The first demonstrations of single-base resolution microchip separations were limited to relatively small DNA fragments.25 With the tendency to increase the number of STR loci analyzed simultaneously for database globalization and with the expansion of the CODIS core loci,26 the number of larger DNA fragments to be separated with a high resolution increases. Liu et al. demonstrated integration of PCR with microchip electrophoresis (ME) using a 14 cm long separation channel on glass microchips.27 While this microchip provided the desired STR profiles, glass microchips of this size and complexity can be prohibitively expensive for wide application. Alternatively, the two instruments that have demonstrated the full sample-in answer-out STR analysis have used either a capillary28 or a 22.5 cm microchannel29 for the DNA separation. This either eliminates the ability to perform the analysis in a single integrated microchip or results in very large, expensive microchip. Therefore, there remains a need to demonstrate fast DNA separations with high resolution for every size of fragments on a small, portable microfluidic chip that can be integrated into a μTAS chip to provide a cost-effective, rapid analysis. In this study, we demonstrate a compact, valveless plastic integrated microfluidic chip run on a prototype instrument that controls multiple functions, including multiplex PCR amplification, pneumatic microfluidic movement, electrophoretic injection and separation, 5-color detection, and data analysis. We demonstrate that, in a small, inexpensive microchip, it is possible to (1) perform integrated PCR-ME to generate profiles for human identification comparable to conventional processes, (2) have the single-base resolution and precision necessary for accurate allele identification in a plastic channel with an effective length of 7 cm, and (3) have a system that can perform consistent separations over 6 months allowing accurate allele calling from a stable binning palette. A microchip that is able to perform an 18-plex PCR followed by a DNA separation with single-base resolution can be rapidly adapted to other assays such as bacterial or STD detection and identification.

■

MATERIALS AND METHODS Chip Design and Instrument. The microfluidic chip is approximately the size of a 96-well plate, 125 mm × 76.5 mm, and contains four identical channels each for a single sample. Thus, theoretically, four samples could be analyzed simultaneously. This chip was fabricated by the microfluidic ChipShop GmbH (Jena, Germany) in a cyclic olefin polymer (COP) plastic. It is injection molded and therefore inexpensive to manufacture. The microfluidic chip (Figure 1A) includes a 1.3

Figure 1. Microfluidic prototype instrument and chip: (A) Image of the chip and identification of key features: the PCR chamber is located in the middle of the channel between sample inlet and sample reservoir, the detection region is at the outlet top of the microchannel; (B) Instrument used to perform the analysis with arrow indicating the chip loading location; (C) Chip−instrument interface that provides pneumatic and electrical connections and optics regions; (D) Side and top view of the sample reservoir with dyed reagents mixing completely after microfluidic movement. Left-to-right PCR products are entering the sample reservoir and mixing with separation reagents for electrophoretic injection.

μL elliptical shape PCR chamber and a separation channel with an injection-to-detection distance of 70 mm. The PCR and separation regions are connected at a sample reservoir via channels that allow fluid flow and mixing of the PCR product with the separation reagents. 8193

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

PCR thermocycling, the microchip was pressurized to 12 PSI. Thermal cycling was performed using noncontact IR-mediated heating and noncontact temperature sensing with the following protocol: 94 °C for 1 min (initial denaturation) and then 27 cycles at 94 °C for 5 s (followed by a ramp cool to 59 °C for 1 min) and 59 °C for 40 s (ramp heat to 94 °C for 1 min between cycles). Last, a final 5 min extension step was performed at 60 °C. These PCR parameters have been chosen following PCR optimization tests described in the Supporting Information (section 3). Following PCR, the microchip was depressurized and positive pneumatic pressure at the sample inlet pushed the PCR products toward the sample reservoir. After the PCR products reached the sample reservoir, they were mixed with the separation reagents by an additional fluidic movement out of the sample reservoir toward the PCR chamber and back in the sample reservoir. Figure 1D shows complete mixing of dyed reagents observed from the side and top of the sample reservoir. The bubble that separated the PCR reagents and the Hi-Di Formamide/GeneScan−500 LIZ Size Standard mixture is able to float to the top of the reservoir and not interfere with the electrophoretic injection. Following the mixing, the detection optics were precisely aligned to the microchannel to account for small differences in channel alignment between runs. The alignment dye was electrophoretically driven through the separation channel to the detection zone from the waste reservoir. While the device has four channels, only one channel could be run at a time due to the optical alignment constraints (mechanical tolerances between optical stacks did not allow for simultaneous alignment of four channels). Thus, this study presents a four channel chip used in only one channel. Further optimization is needed to be able to use the four channels in parallel. Microchip electrophoresis conditions and optimization of the separation and polymer sieving matrix for this short separation length are described in the Supporting Information (sections 4 and 5). Alignment and ME occur in the microchannel with a stage heater set temperature (43 °C). Conventional Process: PCR and Capillary Electrophoresis. Thirty conventional analyses were performed in the forensic laboratory in parallel with the same DNA extracted to allow comparison of the results. Conventional PCR was with a reaction volume of 12.5 μL, using the same primer/reaction mix of the PowerPlex 18 Fast System. The manufacturer’s instructions for thermal cycling were used. Conventional electrophoresis was performed on a 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using the protocol given in Supporting Information (section 6). Allelic Ladder Separations. Ten allelic ladders have been separated in the first laboratory (the same laboratory as the 30 samples, 3 months after these samples), and 10 others have been performed in a second laboratory after an additional 3 month period of time under the exact same separation conditions. The two locations were tested to evaluate whether moving the device impacts performance as conventional capillary systems are typically recalibrated after moving. The preparation was the same as for the PCR-ME except no PCR mix was added and the separation reagents contained allelic ladder. Allelic ladder was added at a volume of 2 or 4 μL for the first and second set of runs, respectively. The change in the concentration was made to be able to easily distinguish the series when overlaid to look at binning palette consistency over time. The allelic ladders were analyzed together to build a binning palette that was used to call the 30 samples. These two

An instrument has been made to interface to the microchip and perform the PCR-ME process automatically. The instrument (Figure 1B) contains all of the hardware required to perform PCR, separation, multicolor detection, and pneumatic fluid movement. The instrument was controlled by a dedicated external laptop. An external air source was required for the pneumatic system in the instrument. The microchip was placed into the chip interface module (CIM) of the instrument, located on the top of the instrument (Figure 1B, white arrow), which aligns the chip features for PCR, separation, and electrical and pneumatic connections. The pneumatic system and electrodes are integrated into the top of this CIM and mate with the chip when the cover is closed. The pneumatic system locks the chip into place and ensures an airtight seal between the instrument and the chip for reproducible pressurization and fluidic movement. Thermal cycling is achieved through noncontact infrared (IR) heating30 and sensing at the PCR region (Figure 1C). The detection region in the microchip separation domain interfaces with confocal optics coupled to an imaging spectrometer. This detection system allows for 5-color detection during DNA separation. Fiber optics provides a robust method for delivering the laser excitation and collecting emitted fluorescence. Data processing for allele calling was achieved using GeneMarker HID software (SoftGenetics LLC, State College, PA, USA) after initial data processing using proprietary software (Lockheed Martin Corp, Bethesda, MD, USA) developed for the instrument. Therefore, after the operator loaded the chip, the remainder of the process, PCR-ME analysis, was performed within the instrument. Integrated PCR-Microchip Electrophoresis (PCR-ME). Reagent Loading into the Microfluidic Chip. The microchips were inexpensive, single-use devices and therefore disposed after each use. Each chip has been previously prepared with a microchannel coating (see protocol in the Supporting Information (section 1)). The separation microchannel was filled by adding 15 μL of separation polymer (4.5% (w/v) hydrophobically modified poly(acrylamide) (HMPAM), Microlab Diagnostics, Inc.) to the outlet reservoir and applying a positive pressure with a syringe. Following polymer loading, 25 μL of separation buffer (1× TTE + 7 M urea) was loaded into the outlet reservoir; 25 μL of polymer was loaded into the buffer reservoir, and 25 μL of polymer was mixed with the 1 × 10−9 M fluorescein in the waste reservoir. Liquid DNA extraction was performed in less than 5 min offchip using conventional benchtop equipment with the prepGEM Saliva kit (ZyGEM corp., Hamilton, New Zealand); see Supporting Information (section 2) for the protocol. PCR amplification mix (Promega Corp., Madison, WI, USA) was prepared off chip before the amplification as described in the Supporting Information (section 2). A PCR mix aliquot was loaded onto the microfluidic chip through the inlet reservoir into the PCR sample chamber. Thirty analyses were performed consisting of 10 independent tests of three different donors. The sample reservoir was filled with separation reagent that consisted of 20 μL of (3:1 volume/volume) Hi-Di Formamide and GeneScan−500 LIZ Size Standard (Life Technologies, Carlsbad, CA, USA). This creates a small air gap inside the channel between the end of the PCR channel and the sample reservoir to prevent reagent mixing prior to PCR. Microchip PCR-ME. Once the chip was inserted inside the CIM, the software driven analysis began. The CIM was first closed by pneumatics. To prevent bubble formation during 8194

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

sets of allelic ladders were run with the same separation conditions as the 30 samples. An additional set of allelic ladders was run at a lower temperature to evaluate the sensitivity of the microchip to changes in separation temperature. Data Processing and Data Analysis. Data processing of the integrated PCR-ME was performed by the proprietary software (Lockheed Martin Corp.) that integrates with SoftGenetics GeneMarker HID software. The GeneMarker software was used to create binning palettes, overlay traces, and perform allele calling. The software will automatically identify the allele peaks. The user can interface with the results if needed, such as recognizing artifacts if the pull-up correction left a residual peak, given that this is a prototype system. Additional optimization of the analysis software for this specific system can be performed in the future. The conventional results were analyzed with GeneMarker HID software for a direct comparison.

■

RESULTS AND DISCUSSION To achieve forensic quality profiles, the PCR and separation subassays were optimized in preliminary studies for the microchip format (ZyGEM extraction of the buccal swabs was performed off-chip). Microchip PCR conditions were developed to achieve the 18-plex amplification with an IR laser and noncontact temperature sensing in approximately one-half the time of a conventional amplification. The annealing and denaturing temperatures were optimized as shown in Figure S1, Supporting Information. Figure S-1A, Supporting Information, shows the average peak height at multiple annealing temperatures for denaturing temperatures of 93 and 94 °C. Figure S-2B, Supporting Information, shows multiple denaturing temperatures with annealing temperatures of 58 and 59 °C. Annealing times between 20 and 50 s were tested with annealing and denaturing temperatures of 59 and 94 °C, respectively. A 40 s anneal time provided strong, balanced profiles and minimized amplification time. The separation conditions were optimized to achieve singlebase resolution in a 7 cm effective channel length. Figure S-2A, Supporting Information, shows that, for this system, joule heating in a 75 μm channel results in a significant loss of resolution. This type of polymer has previously been used on glass chips for DNA sequencing;31 however, further optimization was required for a plastic substrate. The separation channel was subjected to several coating methods to minimize peak broadening (Figure S-2B, Supporting Information). Pretreatment with 1 M NaOH followed by incubation with the sieving polymer was found to provide the highest resolution separation. Previous work with this polymer found that a 4% (w/v) provided the best sequencing results with the range of 2−4% tested. Figure S-2C, Supporting Information, demonstrates that increasing the concentration to 4.5% (w/v) provides slightly higher resolution for fragments below 250 bases with no loss of resolution out to 500 bases. This is likely due to an increase in selectively for the smaller fragments without entering the biased reptation with orientation regime. Therefore, 4.5% (w/v) was used for these studies. Figure 2 shows an example of a microchip (Figure 2A) and a conventional PCR amplification (Figure 2B) of the same donor separated on a conventional ABI 310 Capillary Electrophoresis (CE) instrument for separation and detection. The 1.3 μL amplification can produce a full profile with acceptable quality in terms of peak height and intralocus balance as a conventional amplification when separated on a conventional CE system.

Figure 2. Same donor (donor 3) amplified by (A) microchip and (B) conventional PCR both separated on an ABI 310. The microchip is able to generate profiles with similar peak heights and peak balance as the conventional PCR process.

The interlocus balance of the microchip PCR is lower than the conventional PCR, which may be addressed by further optimization of the thermocycling conditions or the primer concentrations. Moreover, peak broadening appears in the microchip results that has been linked to the chip coating and the polymer composition. Both of these sources of broadening are under investigation for further improvement. The microchip amplification was completed in 45 min compared with 90 min for the conventional method. Ten allelic ladders were separated on the microfluidic system to demonstrate that the system had the resolution and precision required for accurate STR allele calling and ensure that the allele calling was concordant with the conventional process. Figure 3A shows an example of an allelic ladder separation on the microfluidic system. To achieve accurate allele calling, the ultimate goal of this assay is that it must have both high resolution and precise (i.e., low standard deviation) separations. Figure 3 shows the resolution easily allows the identification of alleles differing by two bases and the single-base 9.3/10 peaks of THO1 (Figure 3B). As these are the most challenging peaks to resolve, the resolution requirements for this assay have been achieved. The microchip achieves the resolution necessary for the samples analyzed in this study. Future work with larger data sets with microvariants of the larger molecular weight loci (e.g., FGA) and newer STR kits that may have different resolution requirements can be performed to determine if additional optimization is necessary for this microchip subassay. The microchip allelic ladder separations had a base pair variation of 0.18 bp maximum for all the CODIS loci. This standard deviation is moderately higher than that typically reported in the conventional capillary electrophoresis process, less than 0.10 bp, and close to the forensic target of 0.15. This difference may be due to several factors. First, the polymer solutions used in this study were made on small scales and 8195

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

Figure 3. Allelic ladder separation on the microfluidic platform: (A) a representative allelic ladder obtained on the platform (PowerPlex 18 Fast System) and (B) zoom in the TH01 region, separation of 9.3 and 10 alleles.

small lot-to-lot difference may result in slightly greater standard deviation. As the polymer solution is brought to the commercial manufacturing, these differences may be reduced. Second, temperature control of the separation has been shown to be critical for separation precision in conventional capillary electrophoresis and significant improvements have been made to conventional temperature control systems to improve sizing precision.32 As this is an early prototype system, the temperature control may not be to the standard of conventional instruments that are on their third or fourth generation. Improving this control in future instruments is expected to improve separation precision for the microchip system. Integrated PCR-ME was performed on 30 samples (10 swabs from three different donors) by the microfluidic prototype system and compared with conventional results. The microchips were disposable, single-use devices; therefore, the 30 microchip DNA analyses were performed on 30 different chips with the goal of developing reproducibility data on one channel as the channels are completely independent. The 30 different samples and 20 different allelic ladders of that study have been analyzed in 50 different microchips in the same channel (channel 1). Only one channel has been demonstrated here, but the four channels are working similarly and could be used independently. The processing time from swab-to-result was approximately 2 h including the half an hour sample and chip preparation and the 90 min on chip time. This integrated microchip design allowed the sample to be easily removed from the sample reservoir for additional postanalysis. This provided the opportunity for one to compare the integrated PCR-ME with the same amplified DNA product run on the conventional capillary electrophoresis platform. The 30 samples from the PCR on chip removed from the sample reservoir after the PCRME all generated full genetic profiles that were 100% concordant to the reference samples developed using the same DNA analyzed by conventional PCR/conventional CE. Therefore, the valveless microchip PCR process produced the same result as the conventional PCR in 100% of the runs performed. Moreover, the IR-PCR/ME results shown in this study are representative of the microchip PCR characterized by

that test of IP-PCR/CE integrated with the microchip electrophoresis. To more closely compare the microchip and conventional profiles, intralocus peak balance, a common forensic quality metric, was compared between the two methods. The intralocus peak balances from the 30 microfluidic analyses on the microfluidic prototype system are given in Figure 4, which clearly shows that these ratios are principally greater than 50% for both processes. The intralocus balance ratio mean values were 83%, 87%, and 84% for donors 1, 2, and 3, respectively, on the microfluidic prototype system (Figure 4A). The conventional process yielded in ratios that ranged between 88% and 93% (Figure 4B), with stutter-to-peak ratios less than 15% for both conventional and on-chip processes. While additional optimization of the microchip PCR is required to achieve the same intralocus values as conventional, 75% were above the balance target of 80%. This demonstrated that the low volume PCR (1.3 μL) on a microfluidic chip allows for amplification of STR loci within accepted heterozygous peak balance metrics of this assay. The interlocus balance, calculated as the lowest RFU peak height divided by the largest RFU peak height (considering the homozygous peak heights divided by two or the average of the heterozygous peak heights) across all the loci within a color, was also lower for the microchip amplifications. The microchip amplifications yielded interlocus balances between 0.16 and 0.32 with the conventional method yielding between 0.54 and 0.81. These drops in balance may be due to specific loci being more sensitive to changes in temperature or chemistry conditions. Future studies on chamber size and shape, thermocycling conditions, and PCR chemistry may elucidate the exact cause of these differences and allow even more comparable results. The percentages of allele detected on the integrated microchips for the 10 samples of each of the three donors are given in Table S-1, Supporting Information. The microchip analysis detected 90% of the alleles in mean value compared with 100% of the alleles when analyzed by conventional capillary electrophoresis. Each donor has at least 3 completely full profiles (donor 1:3 full profiles, donor 2:7 full profiles, donor 3:8 full profiles). The donor 1 tests were performed before donor 2 and donor 3. It appeared that donor 8196

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

Figure 4. Intralocus balance calculated for (A) 30 microchip PCR-ME samples and (B) 30 conventional PCR samples.

1 had more missing peaks due to bubbles in the separation channel, possibly introduced by the operator when loading the polymer. Therefore, the improvement in results from donor 1 to donor 3 may be due to an improvement in polymer loading. In forensic laboratories, profiles are uploadable to the CODIS if they include each of the 13 CODIS markers plus Amelogenin. One hundred percent of the conventional profiles had 13 out of 13 CODIS loci plus Amelogenin, and results of integrated PCR-ME would be CODIS uploadable for 73.3% of the samples. While the microchip PCR-ME could provide comparable results to the conventional capillary electrophoresis, there was a difference in performance between the two systems. There are several possible sources of this variation that result in the observed allele dropout. Variations in microfluidic flow of amplified reagents to the sample reservoir may cause differences in microfluidic mixing with the injection reagents or electrophoretic injection causing lower signal of the amplified product. In several cases in which an incomplete profile was generated, bubbles were observed in the microchannels during a postrun inspection. These bubbles may have been due to deviations in fluid flow, manual polymer filling, or small defects in the separation channel that can act as bubble nucleation points. Incomplete profiles often correlated with one of these failure modes, e.g., bubble formation or insufficient transfer to the sample. These issues of bubble formation and fluid flow can be addressed by small design modifications in future work. Figure 5 shows an exemplary set of full profile results from a conventional PCR-CE (Figure 5A), a PCR on chip followed by a conventional CE (Figure 5B), and an integrated microchip

Figure 5. Results obtained for donor 2 with the PowerPlex 18 Fast System: (A) Result from the conventional process: PCR and separation on the conventional instruments, (B) results from a conventional separation of a PCR on chip, and (C) a representative genetic profile, from PCR-ME, obtained on the microfluidic platform for donor 2 with the PowerPlex 18 Fast System. The PP18D STR kit amplifies 18 loci at different locations in the human genome to uniquely identify each person.

PCR-ME analysis (Figure 5C). In the PCR-ME profile, all 18 loci were detected and the alleles were accurately called on the basis of a comparison to the conventional profiles. Baseline subtraction and spectral deconvolution of the 5-color separation was performed in SoftGenetics GeneMarker software. The integrated microchip analysis produces a profile comparable to the conventional process, shown in Figure 5C, provided that the deviations from fluid flow or bubble formation do not occur. A set of allelic ladders was used to create a binning palette for allele calling using GeneMarker HID software. The prototype microfluidic system was able to call 100% of the alleles correctly, even with the 30 microfluidic analyses being analyzed 3 months before this set of allelic ladders. Allele calling accuracy is based on 100% concordance with the conventional method. This was achieved with an effective separation length on the microchip system of only 7 cm, significantly less than the 36 cm capillary length of an ABI 3130xl capillary system Genetic Analyzer or 22.5 cm of another microchannel system29 for STR analysis. 8197

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

Article

These results show that an integrated PCR-ME system can provide results that are 100% concordant with conventional methods and can provide accurate allele calling with the same binning palette over a 6 month period and post-transportation. This stability is critical to the design of the next generation of rapid DNA analysis systems as it demonstrates allelic ladder controls are not necessary on every microfluidic chip. This will allow higher throughput on multichannel systems and opens the possibility of single-channel analysis systems that may be of interest for field deployable rapid DNA analysis systems. This system demonstrates the resolution and precision required for STR analysis in a true compact microchip format that is integrated with PCR. While additional development is needed to make this either a commercial ME-only or fully integrated system, this study represents a critical demonstration of microchip sample analysis with 100% concordance with no loss of allele calling accuracy over a 6 month window or transportation of the instrument.

After 100% allele calling accuracy was demonstrated, the stability of the allelic ladder separation was investigated. Ten additional allelic ladder separations were performed three months after the first set of allelic ladders after transporting the system to another laboratory. The 30 samples analyzed have been performed six months before the last set of allelic ladders and three months before the first set. The separation conditions (e.g., temperature, electric field strength) were the same for each of these sets. No maintenance or recalibration of the system was performed following the 500 km move to the second laboratory to determine if the system is more robust than conventional capillary electrophoresis systems that typically require recalibration. The allelic ladders from this set were overlaid with the original binning palette and the buccal swab samples performed 6 months before. Figure 6 shows the

■

CONCLUSION An integrated PCR-ME microfluidic chip and instrument for human identification by STR analysis has been demonstrated. This microchip performed the STR separation in an effective length of 7 cm, which is significantly shorter than previously described systems. This allows for a small, inexpensive singleuse microchip with the separation performed in less than 15 min. This high-resolution separation was maintained when integrated with microchip PCR. The microchip IR-mediated PCR was completed in less than one-half of the time of a conventional amplification. The IR-PCR of the integrated PCRME device produced more partial profiles than the conventional process, even if some full profiles were obtained; however, all allele calls made were concordant with those produced by the commercially available systems. . Additionally, the allelic ladder binning palette was shown to be stable over 6 months. Taken together, these results provide a significant step for the next generation of point-of-sample-collection forensic instruments that can also be applied to other genetic or clinical applications. The length of the separation channel is a limiting characteristic for many microchip designs. This high-resolution separation allows the chip footprint to be significantly compressed leading to significant cost-reductions in fabrication. The stability of the allelic ladder opens the possibility of not requiring an allelic ladder on every chip or even for an allelic ladder to be run when an instrument is moved to a new location, a critical consideration for field deployable systems envisioned for the future. Future work on optimizing the amplification, instrument, and microchip, such as limiting small variations in stage temperature for the separation and further improving the PCR peak balance, can enhance the quality beyond what was shown here. This system is a critical step to a fully integrated microfluidic system that brings the ZyGEM extraction of DNA into the system along with the other functions such as metering and mixing reagents.

Figure 6. Ten samples overlaid of donor 3 (28, 31.2 at the D21S11 locus) with allelic ladders ran 3 months (under 1000 RFU, lower arrow) and 6 months later (3500 RFU, upper arrow).

traces overlaid for D21S11 loci demonstrating that peaks from each of the separations falls within the bins. This figure also overlays the traces from donor 3 from the PCR-ME study. This donor has peaks at alleles 28 and 31.2 which are shown to be well aligned within the bins and with the allelic ladder peaks. An additional binning palette was made from the allelic ladder separations at each time interval. All loci were again called 100% correctly by the software. This demonstrates that a microfluidic instrument for integrated PCR-ME may be stable for 6 months and after transport even for the stringent allele calling application. The sensitivity of the allele calling to separation temperature changes was tested for the microfluidic system. Allelic ladder separations were performed at a separation temperature 2 °C different from the other studies, and a binning palette was created from these separations. This temperature difference did lead to errors in allele calling particularly for the alleles differing by 2 bases, as D21S11, FGA, or D18S51 (Table S-2, Supporting Information). These errors disappeared when using the appropriate binning palette (Table S-3, Supporting Information), and 100% concordance was observed. The errors in allele calling were reproducible for each donor meaning that each sample had the same incorrect shift in allele call. This is due to the reproducible change in DNA mobility. This sensitivity and size deviation with the temperature is similar to what been described for conventional capillary electrophoresis systems.33 However, this study demonstrated that a microchip system can be sufficiently stable for 6 months and after transportation to achieve 100% allele calling accuracy.

■

ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental protocols, PCR and separation optimization data, and allele detection and calling accuracy data. This material is available free of charge via the Internet at http://pubs.acs.org. 8198

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199

Analytical Chemistry

■

Article

(20) Bienvenue, J. M.; Legendre, L. A.; Ferrance, J. P.; Landers, J. P. Forensic Sci. Int. Genet. 2010, 4, 178−186. (21) Lounsbury, J. A.; Karlsson, A.; Miranian, D. C.; Cronk, S. M.; Nelson, D. A.; Li, J.; Haverstick, D. M.; Kinnon, P.; Saul, D. J.; Landers, J. P. Lab Chip 2013, 13, 1384−1393. (22) Rufer, A.; Aboud, M.; McCord, B.; Gassmann, M. J. Biomol. Technol. 2010, 21, S30. (23) Panaro, N. J.; Yuen, P. K.; Sakazume, T.; Fortina, P.; Kricka, L. J.; Wilding, P. Clin. Chem. 2000, 46, 1851−1853. (24) Fraige, K.; Travensolo, R. F.; Carrilho, E. Braz. Arch. Biol. Technol. 2013, 56, 213−221. (25) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676− 3680. (26) Hares, D. R. Forensic Sci. Int. Genet. 2012, 6, 52−54. (27) Liu, P.; Li, X.; Greenspoon, S. A.; Scherer, J. R.; Mathies, R. A. Lab Chip 2011, 11, 1041−1048. (28) Hennessy, L. K.; Franklin, H.; Li, Y.; Buscaino, J.; Chear, K.; Gass, J.; Mehendale, N.; Williams, S.; Jovanovich, S.; Harris, D.; Elliott, K.; Nielsen, W. Forensic Sci. Int. Genet. Suppl. Ser. 2013, 4, 7−8. (29) Tan, E.; Turingan, R. S.; Hogan, C.; Vasantgadkar, S.; Palombo, L.; Schumm, J. W.; Selden, R. F. Investig. Genet. 2013, 4, 16. (30) Norris, J. V.; Hagan, K. H.; Root, B. E.; Scott, O. N.; Bienvenue, J. M.; Kinnon, P.; Landers, J. P. Demonstration of Rapid LightMediated PCR Amplification of STR Loci Using Polymeric Microfluidic Devices, Presented at the American Academy of Forensic Sciences (annual meeting), Chicago, IL, 2011. (31) Forster, R. E.; Chiesl, T. N.; Fredlake, C. P.; White, C. V.; Barron, A. E. Electrophoresis 2008, 29, 4669−4676. (32) Wheaton, A.; Benfield, J.; Sailus, J.; Felton, A.; Fish, R.; Joe, L.; Lagace, R.; Nordman, E.; Qi, L. The Next Generation Capillary Electrophoresis Instrument for Human Identification, Presented at the International Symposium on Human Identification, 2009; https:// www3.appliedbiosystems.com/cms/groups/applied_markets_ marketing/documents/generaldocuments/cms_093149.pdf. (33) Hartzell, B.; Graham, K.; McCord, B. Forensic Sci. Int. 2003, 133, 228−234.

AUTHOR INFORMATION

Corresponding Author

*E-mail: ph ilippe.d [email protected]. Phone: +33139274287. Fax: +33139274535. Present Addresses ¶

C.R.R.: Signature Science, LLC, 1725 Discovery Dr, Charlottesville, Virginia 22911, United States. # J.M.B.: University of Virginia’s Applied Research Institute, 151 Engineer’s Way, Charlottesville, Virginia 22904, United States. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank the following individuals who contributed to this study: David Saul from ZyGEM Corporation, Teresa Snyder-Leiby from Softgenetics, and Doug Storts from Promega Corporation. Authors are grateful to the DNA fingerprint laboratory of Saint Germain en Laye, France, and to the French National Institute of Scientific Police, Ecully, France. Finally, thanks to Morpho and Lockheed Martin for their financial support and Michael Egan, Darren Albert, and Douglas J. South from Lockeed Martin.

■

REFERENCES

(1) Butler, J. M. Fundamentals of Forensic DNA Typing; Elsevier Science: London, 2009. (2) Vallone, P. M.; Hill, C. R.; Podini, J. M.; Butler, J. M. Forensic Sci. Int. Genet. Suppl. Ser. 2009, 2, 111−112. (3) Vallone, P. M.; Hill, C. R.; Butler, J. M. Forensic Sci. Int. Genet. 2008, 3, 42−45. (4) Giese, H.; Lam, R.; Selden, R.; Tan, E. J. Forensic Sci. 2009, 54, 1287−1296. (5) Wang, D. Y.; Chang, C.-W.; Hennessy, L. K. Forensic Sci. Int. Genet. Suppl. Ser. 2009, 2, 115−116. (6) Easley, C. J.; Karlinsey, J. M.; Bienvenue, J. M.; Legendre, L. A.; Roper, M. G.; Feldman, S. H.; Hughes, M. A.; Hewlett, E. L.; Merkel, T. J.; Ferrance, J. P.; Landers, J. P. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 19272−19277. (7) Horsman, K. M.; Bienvenue, J. M.; Blasier, K. R.; Landers, J. P. J. Forensic Sci. 2007, 52, 784−799. (8) Price, C. W.; Leslie, D. C.; Landers, J. P. Lab Chip 2009, 9, 2484−2494. (9) Christel, L. A.; Petersen, K.; McMillan, W.; Northrup, M. A. J. Biomech. Eng. 1999, 121, 22−27. (10) Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W. K.; Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman, S. H.; Norris, P. M.; Landers, J. P. Anal. Chem. 2003, 75, 1880−1886. (11) Reedy, C. R.; Price, C. W.; Sniegowski, J.; Ferrance, J. P.; Begley, M.; Landers, J. P. Lab Chip 2011, 11, 1603−1611. (12) Zhang, Y.; Ozdemir, P. Anal. Chim. Acta 2009, 638, 115−125. (13) Roper, M. G.; Easley, C. J.; Landers, J. P. Anal. Chem. 2005, 77, 3887−3893. (14) Sinville, R.; Soper, S. A. J. Sep. Sci. 2007, 30, 1714−1728. (15) Wu, D.; Qin, J.; Lin, B. J. Chromatogr., A 2008, 1184, 542−559. (16) Breadmore, M. C. J. Chromatogr. A 2012, 1221, 42−55. (17) Lagally, E. T.; Emrich, C. A.; Mathies, R. A. Lab Chip 2001, 1, 102−107. (18) Liu, P.; Seo, T. S.; Beyor, N.; Shin, K.-J.; Scherer, J. R.; Mathies, R. A. Anal. Chem. 2007, 79, 1881−1889. (19) Pan, X.; Jiang, L.; Liu, K.; Lin, B.; Qin, J. Anal. Chim. Acta 2010, 674, 110−115. 8199

dx.doi.org/10.1021/ac501666b | Anal. Chem. 2014, 86, 8192−8199