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A. Lerner et al. / Soil Biology & Biochemistry 38 (2006) 1188–1192 1189 physical evidence was found in the crime scene. The main suspect, arrested a couple of days later, claimed to have been with the victim on a non-asphalted, nearby parking lot (the alibi area). The suspect washed his clothes and shoes after the murder and the only possible link to the crime scene was a small soil clot (0.2 g) found inside his shoe. 2.2. Soils Soils samples were collected from different locations (Table 1). Soils used for the evaluation of DNA extraction protocols were sampled 5 cm below the surface, and kept on ice until processed. Samples originating from the crime scene were kept in plastic bags on the shelf and in the dark for 6 months until analysis. The soil clot from the suspect’s shoe was not made available for analysis by the court because of the destructive nature of the analysis. 2.3. Soil wash Soil samples in 1.8 ml of 0.05 M buffer phosphate and 0.5% cetyltrimethylammonium bromide (CTAB) were ground using a mortar and pestle. Samples were shaken for 3 h at 4 8C and 200 rev min K1 , and then centrifuged for 10 min at 4 8C and 700 rev min K1 . The supernatant was removed and DNA extraction was performed. 2.4. DNA extraction Five direct methods were used for DNA extraction from bacterial communities: (a) Tsai and Olson (1991) (thereafter ‘T’) with slight modifications: After three freeze-thaw cycles, proteinase K was added to the solution to a final concentration of 50 mg ml K1 and the samples were incubated for 30 min at 37 8C; (b) Zhou et al. (1996) (‘Z’); (c) Yeates et al. (1998) (‘Y’); (d-e) methods based on the commercial kits FastDNA SPIN Kit for Soil (BIO 101, Qbiogene, Inc, Carlsbad, USA) (‘F’) and UltraCleane Soil DNA kit (MO BIO Laboratories, USA) (‘U’). The Y and F methods included a bead beating step. Samples weighing 0.2, 1, 10, 0.6, and 0.25 g were used with methods, T and Y, Z, F, and U, respectively. The weight of the soil samples was the optimum recommended for each protocol. 2.5. Purification of crude DNA extracts The QIAquick Gel Extraction kit (QIAGEN GmbH, Hilden, Germany, and DNA Isolation kit (Biological Ind., Israel) were used to purify DNA. 2.6. PCR amplification One to three microlitre of each DNA preparation from environmental sample were amplified in a PCR reaction mixture (50 ml) using an Eppendorf Mastercycler Gradient (Brinkmann Instruments, Inc., USA). Each PCR mixture contained 0.8 mM of each primer, 0.3 mM of each deoxynucleotide (dNTP), 5 ml of 10! buffer (Promega, Madison, USA), 0.03 unit ml K1 redTaq DNA polymerase (Sigma, Rehovot, Israel), 3.75 mM MgCl 2 ,2ml of10mgml K1 BSA and double distilled, sterilized water to complete the mixture volume. The primers for PCR were specific for conserved bacterial 16S rDNA sequences (Heuer et al., 1997). PCR with primers Gm5f (5 0 -GC-clamp-CCT ACG GGA GGC AGC AG-3 0 ) and 907r (5 0 -CCC CGT CAA TTC CTT TGA GTT T-3) amplified a bacterial 16S rDNA fragment from position 341–928 (Escherichia coli numbering). PCR amplification was performed for 35 cycles as follows: after initial denaturation of 1 min at 95 8C each cycle consisted of denaturation at 95 8C for 20 s, primer annealing at 57 8C for 25 s, and primer extension at 72 8C from 30 s. Cycling was followed by final primer extension at 72 8C from 1 min. PCR products were visualized by electrophoresis in 1% (w/v) agarose gels after 1 mg ml K1 EtBr staining (Sambrook et al., 1989). 2.7. Denaturing gradient gel electrophoresis Strong PCR products of the expected size (550 bp) were subjected to DGGE analysis. DGGE was performed with an Ingeny phor U-2 system (Leiden, The Netherlands). Samples of 43 ml of PCR product were loaded onto 6% (w/v) polyacrylamide gels in 1.0 strength Tris-ethylene-diamineteraacetate Table 1 Properties of the soils used in this study Location Texture Sampling zone Organic matter (%) pH Moisture content (%) Kfar Menachem, Sandy loam (Chromoxererts brown alluvial) Maize rhizosphere 0.9 7.23 5.5–6 Israel Rehovot, Sandy (Haploxeralfs brown–red) Maize rhizosphere 0.5 8.4 27.9 Israel Hula, Israel Peat (Lacustrine gley) Surface soil 29.3 7.26 84.5 Coconut Compost (15% polystyrene) 85 4.8–5.6 30–35 residues medium Crime scene Sandy clay loam (Hamric alluvial soils and gley) Surface soil N.D. N.D. N.D. Alibi scene Sandy clay loam (Calcareous sandstone) Surface soil N.D. N.D. N.D. Suspect’s home Sandy loam Surface soil N.D. N.D. N.D. N.D.: not determined.
1190 A. Lerner et al. / Soil Biology & Biochemistry 38 (2006) 1188–1192 (TAE, pH 8.5) TAE buffer. The polyacrylamide gels were prepared with a denaturing gradient ranging from 30 to 60% (where 80% denaturant contained 7 M urea and 40% formamide). The electrophoresis was run for 20 h at 85 V at 60 8C. After the runs, gels were removed from the set up and stained for 30 min with 2 l of 1!TAE and 100 ml of 10 mg ml K1 EtBr solution followed by washing with 1! TAE for 15 min. The stained gels were immediately photographed using an AlphaImagere System (Labtrade Inc., FL, USA). 2.8. Cluster analysis The analysis was performed as blind test in which the operator was unaware of the origin of the samples. Cluster analysis of profile similarity was performed using the Discovery Series Quantity One 1-D Analysis Software Version 4.4.1, PC (Bio-Rad, Rishon Le Zion, Israel) and UPGMA. 3. Results 3.1. DNA extraction All the protocols used in this study were based on direct DNA extraction. Direct DNA protocols include three main elements: chemical, physical and enzymatic lysis (Miller et al., 1999). Each protocol, which had been tested herein, included one or more of these elements. Of the methods tested, only protocols T, F and Z were successful in extracting DNA from a Rehovot and a Kfar Menachem maize rhizosphere soil. DNA was also successfully extracted from a coconut residues medium and from Hula soil, two highly organic soils, using the T and F methods (Table 1). The use of the DNA Isolation kit (Biological Ind., Israel), failed to remove humic acids and resulted in brownish samples that could not be amplified by PCR using primers for the 16S rRNA gene. After a second purification step, including electrophoretic separation of humic substances from DNA in agarose, DNA excision from the gel and purification using QIAquick Gel Extraction kit (QIAGEN GmbH, Hilden, Germany), PCR products were obtained. As a lesser amount of impurities co-extracted with DNA, protocol T was selected as the DNA extraction method to be used for the evaluation of soil microbial community analysis for forensic purposes. 3.2. Microbial community analysis Soil samples were taken at various places at and around the murder scene, at the alibi area and near the family parking lot at the main suspect’s home. In some forensic cases, the amount of material collected is so low that no replicate samples can be obtained (for example, soil adhering to a sole, or to a piece of cloth). In order to reflect this situation, the presented analysis was performed: (i) without prior knowledge of the origin of the samples, and; (ii) with only one replicate per location. Therefore, to mimic the amount found on the suspect’s shoe (around 0.2 g), which was not available, and to evaluate the efficiency of the protocol T, 0.2–0.6 g of soil was extracted. DNA was retrieved from all samples. In some instances, amplicons were only obtained after dilution of the DNA samples. DGGE was performed on the samples originating from the crime scene, from the alibi scene, from the suspect’s home and from different geographical places in Israel in a blind test. Banding patterns were compared using cluster analysis. All samples collected from the crime scene and surroundings clustered together. All the samples from the alibi scene and surroundings were clearly separated from the crime scene samples. They clustered closer to the samples from Rehovot and Kfar Menahem, two soils with a texture similar to that of the alibi scene samples. Also, a Beit Dagan and a Hula soil, both richer in organic matter appeared to be closer to the crime scene samples. However, a sample from the suspect’s home also clustered with crime scene samples (Fig. 1). 4. Discussion 4.1. Use of soils in forensic science Molecular approaches have become a common tool for the analysis of the effect of plant cover, agricultural amendments, pollutants and environmental disorders on soil microbial communities (Torsvik et al., 1998; Marilley and Aragno, 1999; Smit et al., 2001; Johnsen et al., 2001). Such tools may also prove useful in criminal investigations to link a suspect to a crime scene. The first requirement for implementing such methods is a reliable, convenient and reproducible method of DNA extraction from soils. A number of methods for extracting DNA from diverse environments such as soils are available. Thus, the first step of this study was to compare the efficacy of DNA retrieval with various methods on a particular soil type (Haploxeralfs brown– red of Rehovot). The amount of DNA extracted was evaluated by running the DNA obtained on gel agarose and comparing the band intensity after EtBr staining. Zhou et al. (1996) found a negative correlation between cell lysis efficiency and clay content, in contrast to Ranjard et al. (2000) who found no such significant correlation. Our results are in better agreement with those of Ranjard et al. (1998) as the ‘Z’ protocol (Zhou et al., 1996) applied to an Haploxeralfs brown–red soil with a low clay content only yielded low amounts of DNA. Bead beating, although recommended for sandy soils (Yeates et al., 1998), was found to be rather inefficient for this Haploxeralfs soil. Furthermore, only three out of the five methods evaluated successfully extracted DNA from a loamy sand test soil (Chromoxererts brown alluvial of Kfar Menachem). Further comparison with other soil types clearly showed that the timeconsuming Tsai and Olson (1991) protocol (protocol T) was the most suitable. The combination of physical, chemical and enzymatic attack of the cell wall in addition to initial grinding of the sample enabled efficient recovery of DNA from small samples (0.2 g) and from all soil types. Furthermore, DNA could also be extracted and amplified from pieces of
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