Phillip Belgrader and Michael A. Marino
Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, Rockville, MD 20850

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Blood is a convenient source of DNA, and bloodstain cards are routinely used by our laboratory to collect and store reference samples in a repository for the identification of military personnel by DNA typing. In addition, an increasing number of law enforcement agencies are using the bloodstain cards to establish DNA repositories and databanks of convicted felons and/or sex offenders. As a result, the number of cards being generated is growing rapidly. In order to minimize the man-hours required to perform DNA typing off these cards, the development of high throughput systems for DNA purification, PCR amplification, and detection is essential. We have assembled a robotic system that couples DNA purification from bloodstain cards with PCR amplification in a completely automated and uninterrupted process. This system includes a track-mounted articulated robotic arm, a microplate dispenser, a liquid handling pipetting station, and thermal cyclers with pneumatic heated lids. Robots offer distinct advantages since they can perform routine, monotonous tasks with great speed, accuracy, and duration. This system will enable thousands of samples to be processed a day and is designed to be modular to allow peripheral devices to be added or upgraded as needed. Current work involves using the robotic system to produce PCR amplification products derived from STR loci. To further reduce man-hours, these products are then detected by capillary electrophoresis, which eliminates the requirement to cast a gel and manually load the samples.

STR typing involves manually purifying genomic DNA from blood or tissue, subjecting the purified DNA to PCR to amplify polymorphic STR loci, and separating and detecting the PCR products by slab gel electrophoresis. The entire process is relatively time-consuming, labor intensive, and susceptible to sample switching. Therefore, a variety of new detection technologies for high througput DNA typing are being developed to distinguish polymorphisms in a low cost, rapid, automated manner ^1-10. Capillary electrophoresis (CE) and capillary array electrophoresis (CAE) are attractive replacements for slab gel electrophoresis since runs can be accomplished much faster, and gel casting and manual sample loading are eliminated^1-3. However, to get the full benefit of instrumentation capable of rapid DNA analysis, the rate of sample processing, which includes DNA purification and PCR amplification, must be dramatically increased.

Bloodstain cards are routinely used by our laboratory to collect and store reference samples in a repository for the identification of military personnel by DNA typing. In addition, an increasing number of law enforcement agencies are using the bloodstain cards to establish DNA repositories and databanks of convicted felons and/or sex offenders. As a result, the number of cards being generated is growing rapidly, and the development of a high throughput system for sample processing and analysis is essential. We are developing a robotic system that couples DNA purification and PCR of STR loci in an automated manner, and the resultant PCR products were characterized by automated capillary electrophoresis analysis¹¹.

Samples. Bloodstain cards (Fitzco, Minneapolis, MN) were spotted with blood from six staff members, air dried, and stored at -20°C. Punches (1/16 in) were manually taken from the cards and placed in special 96-well microplates, termed Cycloplates (Robbins Scientific, Sunnyvale, CA), with one punch per well.

Robotic Sample Processing. The robotic system consisted of T265 robotic arm on a 4-meter linear track (CRS Robotics, Burlington, ON), an MF-102 microplate feeding station (Eastern Technical Sales, Manchester, NH), a liquid-handling pipetting station (Rosys, Wilmington, DE), a heat block, two Progene thermal cyclers (Techne, Princeton, NJ) and a microplate holder (Beckman, Columbia, MD). The system was programmed using Robcomm software version 4.3 (CRS Robotics) and RAPL-II programming language (CRS Robotics).

Capillary Electrophoresis Analysis. PCR product was analyzed according to the 310 protocol supplied by Applied Biosystems Division/Perkin Elmer.

In an earlier report, we described a prototype system that coupled DNA purification and PCR using a modified Biomek 1000 robotic workstation¹². This was accomplished by implementing a novel method for purifying DNA on bloodstain card punches. Inhibitors of PCR were extracted from the punches, but the DNA remained tightly immobilized to the punches, and was subsequently amplified by solid-phase PCR. Major advantages to this approach, compared to conventional methods in which the DNA is removed from the punch, is that potential for cross-contamination of DNA between wells of the microplate is eliminated.

The next generation robotic system¹¹ represents a practical approach to perform high-throughput sample processing and represents a major advancement from the original system. It consists of a pipetting station with improved liquid handling, a microplate feeder capable of storing nearly 10,000 bloodstain punches in microplates, and thermal cyclers specifically designed for robotic arm accessibility and interfacing capabilities (Fig 1). A routine was written using Robcomm software and RAPL II programming language to instruct the robotic arm to automatically grab microplates from the microplate feeder and transport them to the pipetting station for DNA purification, the heat block to dry the punches, the pipetting station to perform PCR set-up, the thermal cycler to perform PCR, and finally the microplate holder for storage.

To begin the process, 96-well microplates, each containing 16 bloodstain card punches from six different individuals, were placed in the microplate feeder (Fig. 1, upper panel). Appropriate solutions, PCR mix, and Amplitaq polymerase were loaded into the containers on the pipetting station platform (Fig. 1, lower panel). The PCR mix contained fluorescein-labeled primer pairs to amplify either the TH01 or vWF STR polymorphic locus. The program was initiated via computer and the entire process of DNA purification and PCR was performed in a continuous and automated manner. At the completion of the routine, the microplate containing PCR products was manually removed from the microplate holder, and all 96 samples were analyzed by CE. Electropherograms displayed robust PCR products that were automatically sized by the CE software (Fig. 2). Positive results were readily obtained from all samples, and there was never any indication of sample mixing or contamination. The genotype calling was consistent and accurate, matching the genotypes previously determined for all six individuals by validated, conventional forensic testing.

DNA purification and PCR set-up required 45 min, and PCR required 2 hours. Two additional thermal cyclers are needed to avoid a bottleneck at the PCR step. Capacity with four thermal cyclers are needed to avoid a bottleneck at the PCR step. Capacity with four thermal cyclers would result in 96 samples processed after 2.75 hours, and an additional 96 samples every 45 minutes thereafter. Continuous operation of the system would yield 2400 samples processed and ultimately typed per day. However, utilizing scheduling software to maximize system efficiency and staggering some of the applications (i.e. when a microplate is placed on the heat block, another microplate is transferred from the microplate feeder to the pipetting station) would further increase throughput. In any event, the system would provide a sufficient supply of processed samples at the front-end to effectively utilize fast analysis technologies.

REFERENCES

Figure 1. The robotic system (upper panel) consisted of a robotic arm on a linear track, a plate feeder, a pipetting station, two thermal cyclers, and a plate holder. The pipetting station (lower panel) contained a Perkin-Elmer MicroAmp tube rack for holding a microplate, a custom lid for the microplate, beakers containing One-step reagent (A), TE buffer (B), and ethanol (C), troughs containing PCR mix (P) and liquid wax (W), and Amplitaq polymerase (P) in a 1.5 ml microcentrifuge tube in a rack. Microplates containing bloodstain card punches of six individuals were placed in the plate holder. Each microplate consisted of 16 punches for each individual. The punches were arranged on the microplate as follows: individual 1 (columns 1 and 7), individual 2 (columns 2 and 8), individual 3 (columns 3 and 9), individual 4 (columns 4 and 10), individual 5 (columns 4 and 11), and individual 6 (columns 6 and 12).

Figure 2. Capillary electrophoresis analysis of samples processed using the robotic system. Samples were prepared using either the TH01 or vWA STR primer pair. All samples on the microplates were analyzed. Shown are samples analyzed from microplate positions A1-A6 for TH01 (A) and vWF (B). Solid peaks represent fluorescein labeled PCR-products of the respective locus, and the alleles are indicated by numbers. Open peaks represent the ROX-labeled GS350 DNA size marker. X-axis = base-pair size; y-axis = relative intensity of fluorescence.