The Bio Mek 2000Dna Iqtm system

This system (Figure 3.4) uses the DNA IQTM kit-based chemistry: cells are briefly lysed either in a proteinase K-containing buffer or in the DNA IQTM lysis buffer at either 57°, 68° or 95°C, depending on the substrate composition. For semen

Figure 3.4 The DNA IQTM system (Promega Corporation) with the BioMek® 2000 robot (Beckman Coulter). Reproduced with permission from Promega Corporation

stains differential extractions were performed. Lysates were then centrifuged prior to loading onto the robot. After initial contamination tests showed that a low level of contamination was introduced during the robotic extraction, the software method was modified to include automated resin addition, as well as the use of a 96-well deep-well plate and the replacement of the initial shaking step with a pipetting step (Greenspoon et al., 2004). These changes are now included in the BioMek® 3000 Workstation (McLaren et al., 2006). Greenspoon et al. (2004) have validated the BioMek® 2000/DNA IQTM system by extensive contamination, efficiency and sensitivity tests described below. To detect contaminations a 'checkerboard' test was used: samples containing a concentrated source of DNA were loaded into wells with DNA alternating with reagent blanks across the whole plate, forming a checkerboard pattern.

To assess a system's efficiency at extracting low levels of DNA, a sensitivity study utilizing different dilutions of triplicated bloodstain punches (described below) was carried out: one sample was extracted by the automated procedure, one by the manual DNA IQTM process and one by phenol-chloroform extraction. At dilutions of 1 : 10 and 1 : 100, all three methods produced similar yields of DNA. However, at a dilution of 1 : 1000 there was clearly a better yield from the BioMek® 2000/DNA IQTM method than from manual extraction or from automated extraction using the PowerPlex® 1.1 or 16 BIO systems (Promega Corporation, Madison, USA). For sensitivity studies, blood from two different donors was deposited on various substrates (e.g. hand soap and lotion, carpet, black underwear, blue jeans, contraceptive foam, dirt, canvas). The aim of these different depositions was to study if inhibitory substances from the sample carrier might persist throughout DNA extraction and interfere with binding of DNA to the silica-coated paramagnetic resin. In this study, the STR profile was obtained for all of these depositions except for one donor's sample, which had been deposited on synthetic canvas. It seems likely that this failure resulted from the fact that the pre-heating step at 95°C can melt synthetic material and thus possibly destroyed the sample. However, STR profiles for synthetic canvas deposition were successfully obtained in the case of the second donor, indicating that these deposition conditions did not impose an absolute block to sample extraction; the exact reason for the failure in the first case is not known. DNA was successfully extracted from sexual assault samples, cigarette butts, blood stains, buccal swabs and various tissue samples, with no evidence of contamination throughout the extensive validation studies reported by Greenspoon et al.

(2004). In addition, DNA extractions from manual pre-treated bone, hair and epithelial cells from touch evidence have been validated by Crouse et al.

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