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A. Lerner et al. / Soil Biology & Biochemistry 38 (2006) 1188–1192 1191 (a) 0.44 0.60 0.70 0.80 1.00 Beit Dagan (b) cloth containing traces of soil type (crime scene) and weighing 0.2–1 g. Another advantage of protocol T approach was the relatively low amount of DNA shearing, which increased when bead beating was applied. Shearing may lead to amplification of chimeric products and to bands that do not reflect the actual diversity in a subsequent DGGE analysis (Wintzingerode et al., 1997; Roose-Amsaleg et al., 2001). In addition, more humic substances were co-extracted with bead beating (protocol F) than with protocol T. 4.2. Microbial community analysis Hula valley Rehovot Crime scene Crime scene surrounding area Crime scene Suspect’s home Kfar Menachem Rhizosphere Rehovot Rhizosphere Alibi scene,10m west Alibi scene Alibi scene,10m east Alibi scene,10m south 1 2 3 4 5 6 7 8 9 10 11 12 13 Fig. 1. Cluster analysis (A) and DGGE profiles (B) of bacterial communities from different soils. Lanes: 1, crime scene; 2, Alibi scene; 3, Alibi scene, 10 m south; 4, Alibi scene, 10 m east; 5, suspect’s home; 6, crime scene surrounding area; 7, Alibi scene, 10 m west; 8, Hula valley; 9, Beit Dagan; 10, Rhizosphere Rehovot; 11, Rhizosphere Kfar Menahem; 12, Rehovot, and; 13, CRIME scene. Profiling of bacterial soil and rhizosphere communities by denaturing gradient gels is a powerful tool for rapid analysis of temporal and spatial variations in the soil and the rhizosphere. Although the samples from the suspect’s home and from the crime scene could not be separated based on cluster analysis, DGGE reflected a large bacterial diversity, which, in most cases clustered according to soil type and location. Plant roots are sources for organic matter, and directly influence the bacterial community associated with them, as reflected in changes in PCR-DGGE patterns between bulk and rhizosphere soil samples. In the test case described here, there was partial plant cover on soils at the crime scene and in the garden next to the suspect’s house, but not at the alibi scene. This might have influenced the bacterial composition, yielding more similar banding profiles. The simulation presented here called for the evaluation of non-replicated samples, a situation which can seriously limit the sorting of samples of different origins. We suggest that by replicating the methods used for fingerprinting and the analytical approaches, statistical support could be achieved. In an accompanying paper (Lerner et al., 2006), we show that the profiling of rhizobacterial populations performed with DGGE and different primer sets and with ribosomal intergenic spacer analysis, and analyzed by different statistical methods yielded similar results. Sample collection can have an effect on subsequent analyses and proper handling, including tool disinfection, the use of plastic bags to slow down gas exchange and appropriate and standardized storage conditions are recommended (Wintzingerode et al., 1997). PCR-DGGE is powerful fingerprint tool but it also has drawbacks: for example, bands can migrate to the same position, there is a strong bias for dominant populations, and multiple rrn copies from the same organism can yield different bands (Nannipieri et al., 2003). These biases appear in addition to those generated by differential DNA extraction and amplification (Roose-Amsaleg et al., 2001). 4.3. Recommendations In conclusion, the following should be considered for an in-depth evaluation of the approach: whenever possible, samples should be collected as soon as the crime is discovered, as much material as possible should be retrieved from the locations under investigation, including soils with and without rhizosphere, from different depths and different positions at and near the crime scene, and in unrelated areas; Sample collection and storage should be standardized to minimize their impact on the bacterial community, and the analysis should be performed as rapidly as possible after sample collection (Wintzingerode et al., 1997); Samples should be analyzed using a number of primer sets, including phylogenetically-restricted primers targeting specific populations. Statistical analysis should be performed using at least two different methods such as cluster and principal component analysis. Whenever needed, sequence information (from bands running identically in DGGE for example) should be sought to strengthen statistical power. We also suggest that a molecular survey of soils from different geographical and climatic regions should be undertaken using the guidelines stated above to obtain a large amount of data
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