Can denaturing gradient gel electrophoresis (DGGE) analysis of ...

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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