o extractions based on paramagnetic beads. The systems were tested for a number of output factors, including DNA yield, purity and integrity, and suitability of DNA for downstream applications such as PCR. Anonymized EDTA blood samples and mock samples (water blanks) were used in the testing, and the survey also provided specifications for each system, including throughput, sample volume capacity and costs. Table 3.1 shows a summary of the results. Most of the systems did not have a bar-code system included in their design, so samples had to be tracked manually by means of tube/sample location. A final analysis of the survey results concluded that the integrated systems, which combined dedicated instruments and extraction chemistry, gave significantly better results than those that adapted existing DNA extraction kits to an automated platform using standard liquid-handling robotics. The study further concluded that although these kit-based chemistries could be optimized to a level satisfactory for diagnostic use, such systems would most likely require an extended optimization time to ensure good results. In terms of the different extraction chemistries tested, the solid-phase extractions gave the best results in spite of issues involving blocked wells and insufficient vacuum power, problems that are particular to this methodology. Overall, the simplest and most versatile systems appeared to be those based on paramagnetic bead chemistries.
Adapting a robotic extraction system for forensic casework can be a formidable problem, since the system has to be flexible enough to function efficiently across the enormous range of variation in DNA content obtained from a wide variety of forensic samples, such as blood, saliva, hair, vaginal swabs and contact stains on various carrier materials. The number of cells present in a sample can vary broadly - from more than 1000 nucleated cells in half a microlitre of blood to only a few cells in a sample obtained from a touch or a sneeze. Contact traces are becoming more and more prevalent in criminal cases, so the efficiency of extraction systems has to be rigorously successful down to pico-gram amounts of DNA while remaining exquisitely sensitive to the need to avoid potential contamination events.
Although there were early applications of automated extraction systems for clinical diagnostics, automated extraction systems for forensic evidentiary samples have only recently been reported (Greenspoon et al., 2004; Crouse et al., 2005) with the application of the BioMek® 2000 Workstation (Beckman Coulter, Inc., Fullerton, USA) combined with the DNA IQTMkit (Promega Corporation, Madison, WI, USA) for forensic casework samples. Nagy et al. (2005) have validated the Qiagen BioRobot M48 workstation (QIAGEN GmbH, Hilden, Germany) for nearly all types of forensic samples, and the smaller counterpart of this instrument, the Qiagen EZ1 (QIAGEN GmbH, Hilden, Germany), was shown to be valid for forensics work by Anslinger et al. (2005) and Montpetit et al. (2005). The DNA IQTM system has been automated for the initial testing of forensic samples on robotic platforms such as the Tecan Freedom EVO® 100 (Cowan, 2006) as well as on the Maxwell™ 16 instrument (Bjerke et al., 2006). The Maxwell™ 16 instrument (Promega Corporation, Madison, US; Figure 3.3) operates differently than many other automated DNA purifica-
tion systems: rather than moving liquids from one well to another to carry out the various stages in DNA isolation and purification, the paramagnetic particles (PMPs) are moved from well to well during the purification process by individual magnets and disposable plungers.
Not surprisingly, all these described methods are based on the paramagnetic beads chemistry, which we and others have concluded is the simplest and most versatile system. However, the question of which automation system is best suited to forensic analysis remains open, and we now discuss three systems that are currently the most extensively validated robotic systems for forensics analysis: the Beckman BioMek® 2000, the Qiagen M48 and the Qiagen EZ1. All three systems use an extraction method that comprises the following steps (Figure 3.1c): cell lysis, binding of the DNA to the silica surface of paramagnetic particles in the presence of chaotropic reagents, washing steps to remove impurities and elution of the DNA.
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