Integrated sample cleanup and capillary array electrophoresis microchip for forensic short tandem repeat analysis

Forensic Science International: Genetics 5 (2011) 484–492

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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig

Integrated sample cleanup and capillary array electrophoresis microchip for forensic short tandem repeat analysis Peng Liu a, James R. Scherer b, Susan A. Greenspoon c, Thomas N. Chiesl b, Richard A. Mathies a,b,* a

UCSF/UC Berkeley Joint Graduate Group in Bioengineering, University of California, Berkeley, CA 94720, USA Department of Chemistry, University of California, Berkeley, CA 94720, USA c Virginia Department of Forensic Science, Richmond, VA 23219, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 July 2010 Received in revised form 7 October 2010 Accepted 7 October 2010

A twelve-lane capillary array electrophoresis (CAE) microsystem is developed that utilizes an efficient inline capture injection process together with the classical radial microfabricated capillary array electrophoresis (mCAE) format for high-sensitivity forensic short tandem repeat (STR) analysis. Biotinlabeled 9-plex STR amplicons are captured in a photopolymerized gel plug via the strong binding of streptavidin and biotin, followed by efficient washing and thermal release for CE separation. The analysis of 12 STR samples is completed in 30 min without any manual process intervention. A comparison between capture inline injection and conventional cross injection demonstrated at least 10-fold improvement in sensitivity. The limit-of-detection of the capture-CAE system was determined to be 35 haploid copies (17–18 diploid copies) of input DNA; this detection limit approaches the theoretical limits calculated using Poisson statistics and the spectral sensitivity of the instrument. To evaluate the capability of this microsystem for low-copy-number (LCN) analysis, three touch evidence samples recovered from unfired bullet cartridges in a pistol submerged in water for an hour were successfully analyzed, providing 53, 71, and 59% of the DNA profile. The high-throughput capture-CAE microsystem presented here provides a more robust and more sensitive platform for conventional as well as LCN and degraded DNA analysis. ß 2010 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic human identification PCR cleanup Lab-on-a-chip Microfabrication Capillary electrophoresis Genetic analysis

1. Introduction Driven by escalating backlogs and the challenges of analyzing evidence with a wide range in DNA quantity and quality [1–4], forensic scientists are always seeking techniques that can improve short tandem repeat (STR) analysis for better throughput, cost, sensitivity, and reliability. While process automation has advanced significantly through the application of robotics [5,6] and capillary electrophoresis (CE) [7], the translation of the process into a nanoliter scale in an integrated microfluidic format is more desirable due to its potential for reducing reagent and time consumption, enhancing the sensitivity and reliability, and eliminating the risk of sample contamination and mix-up [8– 11]. Towards this goal, multi-lane microfabricated capillary array electrophoresis (mCAE) system has been developed for high-

* Corresponding author at: Department of Chemistry, 307 Lewis Hall, MS 1460, University of California, Berkeley, CA 94720, USA. Tel.: +1 510 642 4192; fax: +1 510 642 3599. E-mail addresses: [email protected], [email protected] (R.A. Mathies). 1872-4973/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2010.10.009

throughput STR typing [12,13]. The integration of on-chip CE with PCR was successfully demonstrated for real-time forensic human identification [14,15]. Sample processing steps, such as DNA extraction, were also conducted on-chip [16,17]. These achievements validate the feasibility of conducting high-performance forensic STR analysis on integrated microfabricated devices, but further process optimization of integration for routine forensic investigations is needed. The most valuable advantage provided by microfabrication technology is the ability to integrate additional functions into the STR analysis process, which are critical to enhance the performance, but not economical in conventional formats. For example, current STR analysis bypasses a post-PCR cleanup step for routine sample processing in order to save time, cost and excessive sample handling. However, direct electrokinetic injection from high-salt PCR products introduces an injection bias against large DNA fragments and only a small fraction of the PCR products (<1%) are injected for analysis, resulting in reduced sensitivity [10]. Although post-PCR purification and concentration prior to CE analysis improves the sensitivity, the extra time and cost consumption, and increasing risks of sample contamination have limited its adoption in forensic investigations [18–20]. Microfabrication technology

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allows us to explore the integration of PCR sample cleanup with electrophoretic analysis on a single device to overcome the problems of direct electrokinetic injection without introducing additional issues. A variety of on-chip purification, concentration and separation methods have been developed, including membrane filtration [21,22], sample stacking [23,24], and sample extraction [25]. Although these approaches have demonstrated improvements in sensitivity, the delicate operations are incompatible with highthroughput on-chip integration. An oligonucleotide-based gel capture method was developed in our group for DNA sequencing [26] and genotyping [27], but would be difficult to optimize for a high-order multiplex capture process. To address this issue, we have developed an integrated STR sample cleanup and separation microdevice and method that employs a streptavidin capture gel chemistry coupled to a simple direct-injection geometry [28]. STR products having one dye-labeled strand and the other labeled with biotin are efficiently captured and concentrated in a photopolymerized streptavidin gel plug, followed by washing and thermal release for separation. An entire analysis can be complete in about 40 min. Compared to conventional microchip CE with a crossinjector, the fluorescence intensity was improved 14–19-fold for 9plex STR products. However, to make this device and method practically useful and cost-effective for routine forensic work, the scaling of this technology and method to high-density array structures is essential. In this study, we developed a 12-lane capture-capillary array electrophoresis (CAE) microdevice that combines the efficient streptavidin-capture-gel chemistry [28] presented earlier with an improved automated process and the classical design of highthroughput mCAE systems [29]. To evaluate the capture method for high-throughput forensic DNA profiling, we performed 9-plex STR analyses from standard genomic DNA. The fluorescence signals obtained using the capture-CAE device were compared with those using conventional cross injection under the same condition. The limit of detection of this system was also determined for forensic applications. Finally, we tested the ability of the capture-CAE microsystem to process and improve the analysis of touch evidence collected from unfired bullet cartridges that were removed from a pistol submerged in water. This study is a significant step towards the practical application of this integrated capture-separation process in forensic investigation. 2. Materials and methods 2.1. Microchip design The design of the 12-lane capture-CAE microdevice is presented in Fig. 1. On a 4 in. glass wafer, twelve 10-cm-long separation channels sharing a common anode are grouped into six doublets, similar to our previous mCAE devices [12]. Each doublet includes two capture gel inline injectors with two sample wells and one shared cathode and one waste well. The capture gel inline injector is a 500-mm-long double-T channel junction with a tapered structure for PCR product cleanup, concentration, and inline injection. The tapered structure is designed to keep the capture gel in place during gel loading. All features were isotropically etched to a depth of 40 mm and a final width of 160 mm using the same wet etching method as described previously [12]. Prior to use, the microchannels are coated with 0.25% polyDuramide (pDuramide) dynamic coating polymer to minimize DNA absorption to the channel walls and electroosmotic flow during electrophoresis [30]. The coating procedure consists of 1 M HCl incubation for 15 min, DI water flush, and pDuramide incubation for an hour. After treatment, the chips are flushed with water, and then dried with vacuum.

Fig. 1. Schematic of the 12-lane capture-CAE microdevice. (A) A total of 12 electrophoretic separation channels coupled with capture gel inline injectors are arranged on a 4 in. glass wafer. (B) Each doublet includes two capture gel inline injectors and two sample wells sharing one cathode and one waste well. (C) Expanded view of the gel capture region. A constriction was fabricated at the top of the capture region to keep the capture gel in place during the gel loading process. (D) Expanded view of the hyper-turn structure in the separation channels.

2.2. Compact scanner system A photograph of the instrument used to perform analyses is shown in Fig. 2A. The system has dimensions 12 in.  12 in.  8 in., and it can be used as either a bench-top or portable instrument. The instrument contains a 488-nm diode laser (100 mW, Sapphire 488, Coherent, Santa Clara, CA), a confocal optical system with a rotary objective for detecting four different fluorescence signals, pneumatics for control of on-chip microvalves [14,31], four PCR temperature control systems [14,15], and four high voltage power supplies for electrophoresis. A LabVIEW graphical interface (National Instruments, Austin, TX) developed in-house is used to

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filter and collected at a rate of 5000 data points per revolution of the objective using the DAQ board. During operation, a polydimethlysiloxane (PDMS) elastomer well is secured on top of the cathode and waste wells on the chip to create continuous buffer reservoirs. The microdevice is then placed onto the 6 in. heated stage on the top of the instrument and held in place with a Plexiglas manifold and vacuum supplied by the instrument. The manifold contains Pt electrodes that are positioned within the reservoirs on the microchip for the application of high voltages during electrophoresis. Further design details and schematics can be found at http://www.cchem.berkeley.edu/ ramgrp/scanner. 2.3. Streptavidin capture gel preparation A 500 mL streptavidin gel solution, containing 5% (v/v) acrylamide and bis-acrylamide (19:1, Bio-Rad, Hercules, CA), 8 M Urea, 1 TTE (500 mM Tris, 500 mM TAPS acid and 100 mM EDTA), 2 mg/mL streptavidin-acrylamide (Invitrogen, Carlsbad, CA), 0.0006% riboflavin (w/v) and 0.125% TEMED (v/v), is prepared in an opaque 2-mL scintillation vial with Teflon closure (National Scientific, Rockwood, TN) following the method developed previously [28]. To form a capture gel, the photopolymerization solution was first loaded into channels by vacuum. A viscous 5% LPA solution was loaded into each reservoir to stop hydrodynamic flow. Using a UV exposure setup installed on a Nikon inverted microscope (TE2000U, Nikon) and photomask [28], a 500-mm capture gel plug is formed in the double-T channel junctions. The polymerization of each gel plug is complete in 5 min, 12 plugs are finished in 1 h. Un-reacted solution was evacuated out of the channels and replaced with 1 TTE buffer. 2.4. Short tandem repeat typing Fig. 2. The second-generation CAE scanner system. (A) Photograph of the 12 in.  12 in.  10 in. analysis system. (B) Schematic assembly of the four-color confocal detection system with a rotary objective. Design details and schematics will be found at http://www.cchem.berkeley.edu/ramgrp/scanner.

control the system through a NI 6259 OEM multifunction DAQ board (National Instruments, Austin, TX). This system has the flexibility of accommodating one 150-mm diameter wafer in different throughput (12, 24, 48 or 96 channel) configurations [12,32]. With the incorporation of a temperature-controlled stage, pneumatics, and PCR heater control, this system is capable of performing mCAE analysis alone [12], cleanup and separation of off-chip amplified samples [28], and even fully integrated on-chip PCR amplification and STR analysis [14,15]. The optical system is shown in Fig. 2B. The laser beam is reflected by two dielectric mirrors up through a dichroic mirror (Chroma, Rockingham, VT, Z488bpxr). It then enters the hollow shaft stepper motor, is displaced 7 mm by the rhomb, and is focused into the center of the microplate channel. The low mass of the rhomb objective assembly (48 g) allowed us to use a small hollow shaft rotary motor (Empire Magnetics, Inc. U17-7). The clear aperture of the objective was 2.8 mm, which was small enough to pass through the hollow shaft of the stepper motor. Fluorescence collected by the objective returns through the stepper motor shaft and is reflected by the dichroic mirror into a confocal assembly. The light is focused on a 200 mm pinhole with a 20 mm fl achromat lens, and collimated into a 0.7 mm diameter beam with a 5 mm fl achromat lens that enters the 4-color PMT (Hamamatsu H9797 fitted with special sequential dichroic beam splitter optics, 537dclp, 570dc, 595dclp, Z488bpxr, Chroma). The converted electrical signals are processed using a 5-Hz low-pass

For proof of concept, a 9-plex autosomal STR typing system developed previously based on the primer sequences and fluorescence dye labeling scheme used in the PowerPlex1 16 System (Promega, Madison, WI) was employed to test the capture-CAE system [28]. To enable the post-PCR cleanup and inline injection, the unlabeled primers were replaced with biotin-labeled primers (IDT, Coralville, IA). The STR loci included in the 9-plex system are amelogenin for sex typing and 8 CODIS core STR loci (D3S1358, TH01, D21S11, D5S818, D13S317, D7S820, vWA and D8S1179). To facilitate allele calls, a biotin-modified sizing standard was constructed in-house by mixing a series of purified PCR amplicons with different fragment lengths (60, 80, 95, 120, 140, 160, 172, 250, 275, and 350 bp). These fragments are amplified from pUC19, producing products having one strand labeled with ROX and the other labeled with biotin. This sizing standard can be co-captured and separated with PCR products for allele size calibration. Due to the limited amount of the custom-built sizing standards, the crossinjection experiments employed a commercially available sizing standard (ILS 600, Promega). Genomic standard DNA 9947A and 9948 were purchased from Promega (Madison, WI) and diluted in deionized water (DI water) according to the requirements of the studies. The PCR mixture prepared for 9-plex STR typing is comprised of 1 Gold ST*R buffer (50 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 0.1% Triton X-100, 160 mg/mL BSA, 200 mM each dNTP) (Promega), the primer mixture, 0.16 U/mL AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and DI water. All PCR samples were amplified using a PTC-200 thermocycler (MJ Research, Waltham, MA) according to the manufacturer’s protocol for the PowerPlex1 16 kit. In the capture-CAE studies, PCR products are first mixed with Hi-Di formamide (Applied Biosystems) in a ratio of 9–1, and then

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loaded into the sample wells for analysis. When a sizing standard is included, the mixing ratio of PCR products, sizing standard, and formamide is 8:1:1. In the cross-injection experiments, the sample preparation recipe is kept the same, but an extra heating step at 95 8C for 3 min is performed to denature the PCR products prior to loading. 2.5. Touch evidence preparation The touch evidence samples recovered from unfired bullet cartridges were prepared by the Virginia Department of Forensic Science (VDSF) following the procedure described previously [33]. Briefly, bullet cartridges were picked up out of box and loaded into a pistol by a volunteer with no additional or excessive handling of the bullet cartridges (‘‘real conditions’’). This pistol was then submerged in water for an hour. After collection from the submerged weapon, each cartridge was swabbed using the double swab technique [34]. Cartridges were each swabbed with a sterile damp swab containing approximately 40 mL of Type I (ultrapure) water, followed by a second sterile dry swab. Swabs were allowed to air dry before storing at room temperature. DNA samples were extracted following the VDSF BioMek1 2000 Automation Workstation Procedures Manual for Large volume samples. DNA samples with 2.5% Sarkosyl (Sigma, St. Louis, MO), 1 TNE (Tris NaCl EDTA) buffer, 0.39 mmol dithiothreitol (DTT, Sigma), 1.25 mg Proteinase K (Sigma), and Type I water in a 500 mL volume were digested overnight at 56 8C. The cuttings were then placed in spin baskets to collect all of the associated liquid. DNA IQTM lysis buffer with DTT (1.0 mL of DNA IQTM lysis buffer, DTT at 75 mM final concentration) and 8 mL DNA IQTM resin were added to each sample in a 2 mL tube. The samples were vortexed for 30 s, incubated at room temperature for 5 min, then vortexed again for

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30 s. Samples were then purified utilizing the BioMek1 2000 Automation Workstation (Beckman Coulter, Pasadena, CA), following the VDFS BioMek1 2000 Automation Workstation Procedures Manual (http://www.dfs.virginia.gov/manuals/forensicBiology/ index.cfm). DNA quantitation was performed using the Plexor HY System (Promega) on a Stratagene Mx3005PTM Real-Time PCR System (Cedar Creek, TX) according to the manufacturer’s specifications with a recalibration of standard DNA concentrations. The samples were shipped to Berkeley for analysis on the capture-CAE device. PCR amplifications were performed with 4 mL input DNA in 12.5 mL reaction volume using the protocol described above. 2.6. Capture-CAE device operation The capture and separation process illustrated in Fig. 3 is similar to our previous work [28], but has been significantly modified to minimize manual intervention during analysis. Following the photopolymerization of the capture gel plugs in the chip, a separation matrix (5% LPA with 8 M Urea in 1 TTE) is loaded from the anode to the waste and from the cathode to the sample reservoirs to form a matrix-capture-matrix gel sandwich structure in the capture inline injection regions. The tapered structure in the capture region ensures that the capture gel plug will be retained. Off-chip amplified PCR sample is loaded into sample wells and injected through the capture bed under an electric field of 25 V/cm at room temperature for 10 min. PCR products are bound efficiently to the capture gel via the biotin–streptavidin interaction to form a tightly concentrated plug. After capture, an electric field of 25 V/cm is applied from the cathode to the waste reservoirs to wash all the uncaptured sample contents through the gel to the waste for 5 min. The channels above the capture gel are then cleaned by applying the same electric field from the cathode to the

Fig. 3. Fluorescence images of the capture-CAE operation process. (A) A 500-mm capture gel plug containing streptavidin is formed in the capture region by photopolymerization. (B) Off-chip amplified PCR product is loaded into the sample well and injected through the capture bed using an electric field of 25 V/cm at room temperature for 10 min. (C) The captured DNA products are then washed under the same electric field for 5 min. (D) An electric field of 25 V/cm is then applied from the cathode to the sample reservoir to clear unbound sample above the gel. (E) Next, a backwash step is carried out to wash any unbound materials in the separation channel below the gel towards the waste. (F) The microdevice is equilibrated at 67 8C for 1 min to thermally release the fluorescently labeled DNA strands, which are electrophoresed at 250 V/cm towards the anode for separation analysis.

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sample. To clean the separation channels, a backwash step is carried out to wash any unbound materials from the anode towards the waste. Finally, the microdevice is equilibrated at 67 8C for 1 min to thermally release the fluorescently labeled DNA strands, which are electrophoresed at 250 V/cm towards the anode. The overall process can be completed in 30 min. After each run, all the gels and solutions in the chip are pushed out using water and the channels are cleaned using 1 M NaOH and piranha (7:3 H2SO4:H2O2) for 10 min to prevent run-to-run carryover contamination. To plot the STR traces, the four-color fluorescence data underwent baseline and color crosstalk corrections using BaseFinder 6.1.16, and plotted using Microcal Origin. To determine the allele repeat numbers, the four-color fluorescence data are first converted to binary format and appended with proper header information by a custom LabVIEW program. The preprocessed data files are then analyzed for allele calling using the MegaBACETM Fragment Profiler 1.2 (GE Healthcare, Piscataway, NJ) program which performs baseline and color cross-talk correction. 2.7. Cross injection operation The cross-injection analyses are also performed on the same capture-CAE microdevice with no capture plugs and 5% LPA separation gels in all channels at 67 8C. The PCR samples loaded in the sample wells are electrophoretically injected towards the waste reservoir by applying an electric field of 100 V/cm for 60 s while floating the anode and cathode in order to create an injection plug. A separation field of 250 V/cm is then applied between the cathode and the anode to effect the separation. 3. Results and discussion 3.1. Automated high-throughput analysis In a high-throughput capillary array electrophoresis system, human intervention in the analytical process should be completely eliminated. While an integrated capture and capillary electrophoresis system utilizing the strong binding of streptavidin and biotin has been previously developed by our group, the complicated manual operation of this system, which includes three buffer exchanges, is the limiting factor for scaling up to high-throughput system. Several modifications were made here to achieve the automation of the process in the 12-lane capture-CAE system: (i) Separation matrix (5% LPA with 8 M urea) is loaded into both sides of the capture gel and PCR samples are only pipetted into the sample reservoirs. In the previous system, samples filled the entire injection channel on the top of the capture gel plug. As a result, with the previous design, the subsequent replacement of the sample with a washing buffer is required. In the new design, a simple push-back step by applying voltage from the cathode to the sample reservoir is enough to eliminate excess sample in the channels. (ii) Urea is mixed into the capture gel to lower the denaturing temperature of the double-strand PCR amplicons. In the previous system, a formamide washing step with two buffer exchanges was performed to facilitate the complete sample release for separation at 67 8C. By incorporating 8 M urea into the capture gel plug as well as the separation matrix, this washing step is successfully eliminated. (iii) Another function of the formamide washing in the single-lane system was to improve the separation resolution by stacking the sample plug during sample release for separation. We found a similar resolution can be successfully obtained by simply shortening the capture gel plug from 1 mm to 500 mm. Through the modifications described above, the on-chip STR cleanup, capture and separation system has been successfully scaled up to a 12-lane system and the entire process does not

require any manual intervention. The total time for the captureCAE process is less than 30 min, including 20-min post-PCR purification and <10-min separation. Compared to the off-chip post-PCR purification methods, such as Qiagen MinElute, which has a 30-min procedure with multiple manual operations and centrifugations [18], our on-chip capture-CAE system is rapid, easy to operate, and more reliable due to the elimination of human intervention which may cause contamination or sample mix-up. To test the chip design and the operational protocol for highthroughput forensic STR typing, we analyzed 9-plex STR samples amplified from 50 copies of 9947A standard genomic DNA on the 12-lane capture-CAE microdevice. The same amplified sample was loaded into all 12 sample reservoirs and analyzed simultaneously. As shown in Fig. 4, STR profiles were successfully obtained from all the 12 lanes in 30 min. The average percentage standard deviation of the allele signal-to-noise (S/N) ratios is 18.8% and the average standard deviation of the allele migration time is 9.8 s. The variations are similar to those found in conventional chip-based mCAE separations, and can be effectively corrected by incorporating sizing standards [29]. These results demonstrate that the current chip design and operational protocol are compatible with high-throughput integrated STR sample cleanup and analysis. The average separation resolution of the THO1 allele 8 and 9.3 (7 bp difference) was also calculated to be 3.2  0.1. It is possible to detect the rare 1-bp variants (such as THO1 9.3/10) using the current chip design. Furthermore, the resolution can be improved by simply extending the length of the separation channels to 15 cm. 3.2. Comparison with cross-injection method We also conducted a comparison study between conventional cross injection and the capture inline injection under the same conditions to evaluate the sensitivity improvement provided by the capture-CAE microsystem. Since the design of the 12-lane capture-CAE device is similar to the mCAE system, both the capture inline injection and the conventional cross injection can be performed on the same microchip, which allows us to directly compare these two methods. In this study, the STR samples amplified from 50 copies of 9948 standard genomic DNA were analyzed using both methods. As shown in Fig. 5, the allele S/N ratios in the 9-plex STR profile obtained using the capture inline injection were improved 12.1  1.8-fold over the cross injection. Similar peak balance across the DNA profile was maintained for both analysis methods. The excess primers observed in the cross-injection separation profiles were completely removed by the purification process, demonstrating the effectiveness of sample cleanup, concentration and inline injection. 3.3. Limit of detection A sensitivity study was carried out to evaluate the limit of detection (LOD) of the capture-CAE microsystem by using 9-plex STR samples amplified from serially diluted 9947A standard genomic DNA. Fig. 6A plots the percent allele detection as a function of haploid copy numbers of the DNA template and the S/N ratios of each allele at the 9 loci for the 35-copy samples are shown in Fig. 6B. All 13 expected STR alleles were successfully and reproducibly detected with as few as 35 haploid copies of DNA for each locus (105 pg template DNA). The average S/N ratio is 30.0 and the lowest is 5.0 from X allele in amelogenin. This sensitivity is slightly lower than the previous single-lane system (25 copies) due to the change of the sample loading method. Since the samples are only loaded into the sample reservoirs instead of the entire injection channels, the injection into the capture region is less efficient. Nevertheless, the limit of detection is still improved

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Fig. 5. Comparison of STR profiles obtained using the capture inline injection and the conventional cross injection methods under the same conditions. STR samples are amplified from 0.15 ng of 9948 standard DNA using the 9-plex STR typing system. (A) The top trace was obtained by cross injection using the capture-CAE device. The bottom trace was generated on the same device using the capture inline injection procedure. (B) Graph of the S/N ratio improvement on each allele. The average improvement by using the capture inline injection over the cross injection is 12.1  1.8-fold.

Fig. 4. (A) STR profiles of 0.15-ng standard DNA obtained from the 12-lane captureCAE microdevice. The sample cleanup, concentration, and CE analysis were finished within 30 min. All the traces from the 12 lanes are plotted on the same signal intensity scale. The average percentage standard deviation of the allele signal-tonoise (S/N) ratios is 18.8% and the average standard deviation of the allele migration time is 9.8 s. (B) Expanded view of one of the 9-plex STR traces. The dramatically reduced primer peaks show the effectiveness of the sample cleanup procedure which eliminates the their interference with smaller STR fragment detection.

the copy numbers of each chromosome present in the PCR reaction conform to the Poisson distribution, resulting in a variation of the PCR amplification of each allele [36]. To determine the contribution of template loading variation to the limit-of-detection results, the fraction of the DNA profile as a function of input DNA copy number was calculated theoretically based on the expectation from the Poisson distribution. The probability that an allele can be detected by the system at a given input DNA copy number can be expressed using the Poisson cumulative probability function shown in Eq. (1): Pðx; lÞ ¼ 1 

x1 l i X e l i¼0

significantly compared to commercial CE instruments and crossinjection based microchip platforms (50 copies) [12]. When doing a limit-of-detection study in the low-copy-number (LCN) region (100 pg or 33 copies of each locus) [1,35], one challenge is the occurrence of stochastic effects, which include heterozygote imbalance, stutter, and drop-out of one or both alleles during PCR amplification. One of the primary factors causing these stochastic effects is template loading variation: when a low concentration of genomic DNA is introduced into a PCR reaction,

i!

(1)

where l is the input copy number of each locus (0.5l for heterozygous alleles), e is the base of the natural logarithm, and x is the system detection limit expressed in copy number [37]. The average fraction of the full DNA profile is determined by the average peak numbers obtained and divided by the total allele number, as shown in Eq. (2): P P¼

Pi  100% nt

(2)

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Fig. 6. The limit-of-detection test of the capture-CAE microsystem. (A) Fraction of STR profiles obtained using the capture-CAE device for 9947A standard DNA as a function of input DNA copy number. Full STR profiles were reliably obtained from as low as 35 copies of DNA template. The solid line shows the theoretical fraction of 9-plex profiles fits to the experimental data. The dashed lines are the individual Poisson distributions for detection limits of 1, 3, 8, and 15 haploid copies. (B) Average allele S/N ratios for the 35-copy DNA sample on the captureCAE device.

P where Pi is the sum of the probabilities of detecting all the alleles and nt is the total number of alleles analyzed. The sensitivity of the instrument is 20 pM for FAM, 50 pM for JOE, and 100 pM for TMR (data not shown), which translates to approximately 2 pM FAM, 5 pM JOE, and 10 pM TMR before capture gel concentration. Since one single template generates about 108 DNA fragments in an on-chip monoplex PCR [38], it is reasonable to assume that 107 DNA fragments will be generated for each allele in a 9-plex STR amplification which may have lower PCR efficiency due to reagent competition. Therefore, in a 25 mL reaction volume, the detection limits of the capture-CAE system are estimated to be 3 copies of DNA template for FAM-labeled, 8 copies for JOE-labeled, and 15 copies for TMR-labeled alleles. As shown in Fig. 6A, the experimental detection profile vs. DNA concentration corresponds well with the various theoretical detection profiles, demonstrating that the statistical loss of template does play a significant role in determining the fall-off from 100% profile detection at the lowest concentrations. The individual detection profiles for detection limits of 1, 3, 8, and 15 copies were also plotted vs. input copy number for comparison.

Fig. 7. Analyses of three touch evidence samples recovered from unfired bullet shells which were submerged in water for an hour. In a 12.5-mL PCR reaction volume, 4–5 copies of DNA template were amplified using the 9-plex STR typing system. Top traces in each sample were generated using the cross-injection method on the capture-CAE device – no peaks were obtained. In contrast, the lower traces show that 52.9%, 70.6%, and 58.8% of the alleles can be successfully detected using the sample cleanup, concentration, and inline injection procedure on the same chip. Biotin-labeled sizing standards, shown in red (bottom traces), were co-captured and electrophoresed to facilitate the calculation of allele repeat numbers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Alleles obtained and called using the capture-CAE system. Sample

D3

Expected alleles Shell case 1 Shell case 2 Shell case 3

16, * *

TH01 17 * *

6, * *

D21 9.3 *

29, *

*

D5 30 *

12, *

13 * *

D13

D7

Amel

13 * * *

10,

11

*

*

X, * * *

vWA Y * *

17, * * *

D8 18 * * *

13, * *

Percent profile (%) 14 53 71 59

(*) Allele above calling threshold. *Possible drop-in allele with a repeat number of 32.

We have demonstrated that LCN analysis using the capture-CAE system approaches the theoretical limits determined by Poisson statistics and our spectral sensitivity. The plots in Fig. 6A show that an improvement in our sensitivity by a factor of 3 as well as the use of energy-transfer dye labeling (which eliminates the sensitivity variation between different dyes) [39] would enable nearly full profile detection at 10 input haploid copies, but beyond this limit purely statistical effects that are independent of detection sensitivity preclude the possibility of obtaining a full STR profile. These statistical effects are best circumvented by moving towards digital or discrete single cell analysis methods that guarantee full genomic content [40]. 3.4. Touch evidence analysis A DNA profile may be obtained by swabbing items that have been handled by a suspect, even in the absence of visible evidence [33,41,42]. The ability to obtain this touch DNA evidence has evolved over recent years, and has become a routine practice in forensic investigations in numerous countries around the world. Although touch evidence does not always result in low copy number or low template DNA, the limited quantity of DNA is one of the greatest challenges faced by forensic scientists. Needless to say, an instrument with enhanced sensitivity, but also with integrated functionality that reduces the threat of contamination or sample mix-up, will benefit the DNA analysis of touch evidence or LCN samples. To test the capability of the capture-CAE system for low-copynumber DNA analysis, we analyzed touch evidence provided by the Virginia Department of Forensic Science. In total, three samples, each of which was extracted from a single unfired bullet cartridge that was touched by the same person and retrieved from a pistol submerged in water, were amplified separately and tested on the capture-CAE system. Sample concentrations were determined using real-time PCR (4.1, 4.6, and 4.8 pg/mL) to guide the input DNA quantity for the following PCR amplification. 9-plex STR products were amplified from each sample by loading 5 copies of DNA template in 12.5 mL reaction volumes (equal to 10 copies in 25 mL). As demonstrated in Fig. 7, 53%, 71%, and 59% of the 9-plex STR profiles were successfully obtained by using the sample cleanup, concentration and analysis method on the capture-CAE device. Table 1 summarizes the allele calls of these three samples together with the expected alleles. The average fraction of the DNA profile detected is 61%, which is in accordance with the LOD study described above. With the aid of biotin-labeled sizing standards, these alleles were successfully recognized and called for their repeat numbers. Possible drop-in alleles as well as amplification artifacts were observed in this study due to the LCN and compromised sample quality. As a comparison, the same amplified samples were analyzed using the cross injection method under the same experiment settings, revealing consistent blank profiles. The DNA yields of the unfired cartridges from the control, unsubmerged pistol were approximately 3 times that of the cartridges retrieved from the submerged pistol (17.9  22.6 pg and 5.9  8.1 pg, respectively; n = 15), demonstrating the extremely challenging nature of these samples (unpublished data, S. Green-

spoon). This study dramatically validates the advantages of the capture-CAE system for the analyses of ‘‘touch’’ or low-copy-number/ low template DNA analysis. 4. Conclusions We have successfully demonstrated a 12-lane capture-CAE microsystem that employs a photopolymerized streptavidin-gel capture process in a high-throughput format for rapid purification, concentration and separation of biotin-modified STR amplicons. The process of analyzing 12 STR samples required no manual intervention and can be completed in less than 30 min. The signal intensity has been improved at least 10-fold compared with conventional microchip CE with a cross-injector, allowing the detection of full 9-plex STR profiles from as few as 35 copies of input DNA. This enhanced sensitivity enabled the analysis of touch evidence from unfired bullet cartridges retrieved from a pistol submerged in water. The capture-CAE microsystem hence provides a reliable and robust platform for forensic STR typing of LCN and degraded DNA due to its seamless integration of analytical steps, automated operation process, and 100% efficient sample analysis. This system has the potential to become a routine way to reliably analyze DNA samples in forensic investigations. In the future the development of UV exposure systems for rapid photopolymerization at multiple spots would speed up the formation of gel capture plugs and enable even higher throughput capture-CAE devices. Additionally, this inline capture injection process can be incorporated into an on-chip PCR-CE system, leading to the realization of a highly sensitive, contamination-free fully integrated system to explore more challenging studies in forensics, such as STR typing from single cells. Acknowledgements We thank Samantha Cronier and Brian S. Cho for providing biotin-labeled sizing standards. This project was supported by Grant No. 2007-DN-BX-K142 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice. RAM discloses a financial interest in IntegenX, a company that is developing elements of the technologies presented here. References [1] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40. [2] J.M. Butler, Y. Shen, B.R. McCord, The development of reduced size STR amplicons as tools for analysis of degraded DNA, J. Forensic Sci. 48 (2003) 1054–1064. [3] P. Gill, R. Sparkes, R. Pinchin, T. Clayton, J. Whitaker, J. Buckleton, Interpreting simple STR mixtures using allele peak areas, Forensic Sci. Int. 91 (1998) 41–53. [4] R.A. Wickenheiser, D.N.A. Trace, A review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact, J. Forensic Sci. 47 (2002) 442–450. [5] S.A. Greenspoon, J.D. Ban, K. Sykes, E.J. Ballard, S.S. Edler, M. Baisden, B.L. Covington, Application of the BioMek (R) 2000 laboratory automation worksta-

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