The Qiagen BioRobot M48 (Figure 3.6) is a dedicated DNA extraction instrument that was designed with its own integrated MagAttract® DNA extraction chemistry. This system can handle six samples in 15 minutes, and up to 48 samples in 2 hours. The workstation is equipped with special anti-contamination features, such as a completely enclosed robotic deck, a drop-catcher (as well as a control check for closing the door and cleaning the catcher), filter tips, a flat stainless-steel area and a UV sterilization system for decontamination between runs. After loading the instrument, there are no manual steps up until the point of collecting the pure DNA sample (Figures 3.6 and 3.7). The instrument is equipped with an interface that provides step-by-step instructions for
the run set-up as well as options for sample data import and export. Nagy et al. (2005) have worked out a simple, manageable manual protocol for the pre-treatment of forensic samples combined with an extraction protocol (using the Genomic DNA-Blood and Cells-Standard protocol) that is suitable for the full range of forensic specimen (blood samples, buccal swabs, blood stains, vaginal swabs, hairs, cigarette butts, bone meal, etc.). In their recommended standard lysis protocol, the forensic specimen was completely covered with detergent lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 100 mM Na2EDTA pH 8.1, 1% SDS) and incubated for 15 minutes at 90°C with thorough mixing. The only specimens for which modifications were necessary were telogen hair, sperm and bone meal. For telogen hair (i.e. hair that is in the dormant part of its growth cycle), TNca buffer (10 mm Tris-HCl pH 8.0, 100 mm NaCl, 1 mm CaCl2, 2% SDS, 39 mm DTT, 250 |g/ml proteinase K; Hellmann et al., 2001) was used instead of detergent lysis buffer. For sperm, differential extraction was not used (Figure 3.2c): 20 |l of 0.1 m DTT was generally added to 500 |l of detergent lysis buffer in the presence of sperm cells. For bone meal, lysis was preceded by multiple decalcification steps with 0.5 m EDTA followed by a last precise centrifugation step. Using these sample-specific protocols, complete DNA profiles were even obtained from most different types of bones ranging from 2 to 16 years old (macerated and high-density bones included), as well as from 9-year-old teeth. The only sample for which a profile was not obtained was the macerated skeleton part, for which the analysis resulted only in a positive amplification of the amelogenin system. Results showed that PCR inhibitors were efficiently removed from all of the forensic samples, and the prepared DNA remained stable after 2 years of storage. The BioRobot M48 preparative technology system was evaluated by Nagy et al. (2005) on the basis of a defined cell number model so that the DNA recovery rates could be exactly determined. In a 'real-world' analysis using unknown samples, the M-48 BioRobot® workstation has now been used in our laboratory for the extraction of DNA from more than 40 000 routine laboratory samples with a daily monitoring of the extraction efficiency. In addition, there has been no evidence of cross-contamination so far, as examined by PCR testing using 28-30 cycle reactions.
QIAGEN now offers automated DNA extraction kits specifically designed for both of their robotic systems (the EZ1 DNA Tissue Mini kit and the M48 MagAttract® DNA Mini kit). The company also offers protocols that enable processing of solid material directly in the sample tube with no need for cen-trifugation steps, so that high-quality DNA may be purified from a variety of forensic casework samples (QIAGEN News, 2006, e2). Specific pre-treatment protocols for the DNA extraction from various forensic samples are included.
In conclusion, the precision, sensitivity and reliability of the extraction systems discussed here have been extensively validated for application to forensic casework, and results demonstrate that these systems can produce DNA of high quality such that potential PCR inhibitors are removed and there is no need for any further purification steps. Overall, the automated DNA extraction systems discussed here have been shown to carry out safe and successful extractions for practically all types of forensic sample evidence, and should considerably simplify the reproducible processing of large numbers of samples.
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