Journal of Microbiological Methods 75 (2008) 19–24
Contents lists available at ScienceDirect
Journal of Microbiological Methods
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Towards a universally adaptable method for quantitative extraction of high-purity
nucleic acids from soil
Derek Peršoh a,⁎, Susanne Theuerl b, François Buscot b, Gerhard Rambold
a
b
a
Lehrstuhl für Pflanzensystematik, Universität Bayreuth, Universitätsstraße 30 NW I, D-95440 Bayreuth, Germany
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Theodor-Lieser-Straße 4, D-06120 Halle (Saale), Germany
A R T I C L E
I N F O
Article history:
Received 13 February 2008
Received in revised form 22 April 2008
Accepted 22 April 2008
Available online 14 May 2008
Keywords:
DNA
RNA
Nucleic acid extraction
Soil
A B S T R A C T
A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is
presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic
substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By
the approach described here, the humic compounds are precipitated prior to cell lysis with Al2(SO4)3, and
thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable
content and range of humic acids and should therefore be applicable for a wide spectrum of soil types.
Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for
quantitative comparisons.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
In the course of a joint research project focusing on the diversity of
fungal and bacterial organisms in relation to processes involved in the
nutrient cycling in a spruce forest soil, the basal problem of a
quantitative extraction of nucleic acids was encountered. Recently
published and re-evaluated protocols (see LaMontagne et al., 2002;
Lakay et al., 2007; Weiss et al., 2007) did not provide nucleic acids at
satisfactory purity or quantity from our soil samples.
A major problem with the extraction of nucleic acids (DNA and
RNA) from environmental samples (i.e. soil, compost, and sediments) is the presence of humic substances. Because of their
chemically similar properties to nucleic acids, humic compounds are
not removed during standard extraction procedures (Holben 1994;
Zhou et al., 1996; Moreira, 1998; Bruns and Buckley 2002). Since coextracted humic substances interfere in most manipulations applied
for DNA and RNA analyses (e.g., Tsai and Olson 1991; Tebbe and
Vahjen 1993; von Wintzingerode et al., 1997; Rochelle, 2001; Fortin
et al., 2004), i.e. enzymatic reactions (PCR, transcription, restriction)
and hybridizations to reference nucleic acids, their removal is
essential. Different soil types are characterized by a different
composition and content of humic substances, which makes
necessary to optimize specific protocols for each given soil (Weiss
et al., 2007), a time-consuming and difficult task. Moreover, results
gained by protocols specifically adapted to individual soils are not
necessarily comparable, as different extraction protocols have been
⁎ Corresponding author. Tel.: +49 921 55 2456; fax: +49 921 55 2567.
E-mail address: [email protected] (D. Peršoh).
0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2008.04.009
shown to produce different results (LaMontagne et al., 2002; Carrigg
et al., 2007).
Our aim was to develop a time and cost efficient protocol for the
simultaneous extraction of high-purity DNA and RNA from soils. In
anticipation of comparative studies of different environmental
samples, the optimized protocol had to be universally applicable.
2. Materials and methods
2.1. Experimental site and sampling
The sampling site is located in Solling, a mountainous plateau with
an elevation of about 500 m above sea level near Uslar (Lower Saxony,
Germany), in the experimental area (51 31′N, 9° 34′E) of the ‘Solling
roof project’ (Bredemeier et al., 1998). The field-scale roof experiment
was established in 1989 in a 57-year-old Norway spruce plantation,
growing on strongly acidic Dystric Cambisol (FAO classification) with a
moderate podzolized A horizon.
Sampled soil cores were divided into two litter layers (Of and Oh)
and two mineral soil horizons (Ah and Bv). The samples were
transferred to sterile 15 ml reaction tubes in the field and immediately
stored in liquid nitrogen for transport. In the laboratory, the samples
were subdivided in 0.5 g portions and stored in 2 ml screw cap tubes at
−80 °C until further processing.
2.2. Nucleic acid extraction
2.2.1. Preliminary studies
Comprehensive preliminary tests (more than 300 nucleic acid
extractions) were conducted, the results of which are not presented
20
D. Peršoh et al. / Journal of Microbiological Methods 75 (2008) 19–24
here in detail. Briefly, all nucleic acid extractions were performed
using bead beating (FaastPrep™ Instrument, Bio 101), a step
consistently reported this as being the most effective method in
comparative studies (Kuske et al., 1998; Yeates et al., 1998; Lakay et
al., 2007). The efficiency of extraction reagents that previously lead to
satisfactorily results (Kramer and Singleton, 1992; Griffiths et al.,
2000; Hurt et al., 2001) was analyzed by adding the reagents to 0.5 g
of soil, followed by bead beating, purification of the extract with
phenol:chloroform:isoamylalcohol (25:24:1) and chloroform:isoamylalcohol (24:1), and subsequent isopropanol precipitation. The resuspended brown precipitates were not further purified, but the
extracted nucleic acids were analyzed by visible comparison of EtBrstained bands after agarose gel electrophoresis. Subsequently,
established methods for removing humic substances (Mendum et
al., 1998; Fortin et al., 2004; Dong et al., 2006) were pre- or
appended to the “crude” extraction step. The efficiency of these
methods was evaluated according to the absorbance ratio A260/230 of
the extracted nucleic acid solution.
The preliminary protocol was refined for providing optimal results
with the best suited purification method, which was based on
flocculation of humic substances with Al2(SO4)3, prior to the nucleic
acid extraction.
A test series was conducted to estimate the amount of humic
substances which may be precipitated with a given concentration of
Al2(SO4)3. Different volumes of 0,2 M Al2(SO4)3 solution were added to
solutions of 1 mg pure humic acid (Roth) in 1 ml of water, thoroughly
mixed, and centrifuged at 11,000 g for 1 min. A clear supernatant
indicated complete precipitation of the humic acids.
2.2.2. Determination of the required Al2(SO4)3 quantity
To quickly estimate the Al2(SO4)3 concentration required to
precipitate the humic substances, we used the following procedure.
Five subsamples of a soil sample were treated as described in steps 1
to 4 of the protocol below, with volumes of 0.2 M Al2(SO4)3 solution
ranging from 50 to 250 µl for samples of the mineral soil horizons and
from 200 to 600 µl for samples of the organic litter layers. 800 mg of
glass beads (0.5 mm in diameter) were added, and the mix was shaken
in the bead beating instrument at 5.5 m/s for 1 min. The pH was
adjusted to 8 or above by stepwise addition of 4 M NaOH. The samples
were mixed again at speed 5.5 m/s for 15 s in the bead beating
instrument and the minimum Al2(SO4)3 concentration needed to
produce a clear supernatant after centrifugation (11,000 g for 1 min at
room temperature) was noted.
Subsequently, three series (3 to 5 subsamples each) of nucleic acid
extractions were conducted for each sampled litter layer and soil
horizon. Al2(SO4)3 concentrations covering the range of about 75% to
125% (mineral horizons) and 70% to 90% (litter layers) of the
determined concentration for the respective sample were added to
the subsamples of each series. [For organic samples with higher humic
substance content a relatively lower Al2(SO4)3 concentration proved to
be adequate, probably because in these samples more organic material
is enclosed in intact cell and tissue residues, which does not compete
against the free humic substances in the substrate as long as beating
has not been applied.]
2.2.3. Extraction protocol
1)
2)
3)
4)
5)
6)
7)
8)
Weight 0.5 g of the sample in a 2 ml screw cap reaction tube;
Add 100 µl of 1 M Tris–HCl buffer (pH 5.5);
Add V1 µl of sterile distilled Water (dH2O⁎) // V1 = 900 µl-V2;
Add V2 µl of 0.2 M Al2(SO4)3 // V2 depends on humic substance
content of the sample (see below);
Shake in bead beating instrument (BBI) at 4.0 m/s for 15 s;
Add 1/3⁎V2 µl of 4 M NaOH;
Add V3 µl of 0.1 M Tris–HCl [pH 8] // V3 = 1300 µl-(4/3⁎V2)-V1;
Shake in BBI at 4.0 m/s for 15 s;
9) Adjust pH to 8 // stepwise add 10 µl of 4 M NaOH and shake in
BBI at 4.0 m/s for 10 s until pH is 8 or above;
10) Centrifuge 2 min at 3500 g and room temperature;
11) Carefully discard supernatant with a pipette and note the
volume (V4);
12) Add V5 µl of 0.1 M Tris–HCl [pH 8] // V5 = V4-650 µl;
13) Add 0.5 g of 0.5 mm, 0.3 g of 0.1 mm glass beads and one 4 mm
glass bead;
14) Add 325 µl of extraction buffer (0.4 M LiCl, 100 mM Tris–HCl,
120 mM EDTA, pH 8);
15) Add 325 µl of 10% SDS (pH 8);
16) Shake in BBI at 4.0 m/s for 30 s;
17) Incubate on ice for 1 min to avoid overheating;
18) Shake in BBI at 5.5 m/s for 30 s;
19) Incubate on ice for 5 min;
20) Shake in BBI at 5.5 m/s for 30 s;
21) Centrifuge 1 min at 11,000 g and 4 °C;
22) Transfer 750 µl of the supernatant into a 1.5 ml reaction tube;
23) Add 750 µl of phenol:chloroform:isoamylalcohol (25:24:1);
24) Incubate 5 min on ice and shake well every minute;
25) Centrifuge 15 min at 16,000 g and 4 °C;
26a) Transfer the supernatant into a new 1.5 ml reaction tube;
27a) Add 1 volume, equal to the transferred supernatant, of chloroform:isoamylalcohol (24:1);
28a) Centrifuge 15 min at 16,000 g and 4 °C;
29a) Repeat steps 26 to 28 once;
30a) Transfer the supernatant to a new 1.5 ml reaction tube;
31a) Add 0.1 volume of 5 M NaCl and 0.7 volumes of isopropanol;
32a) Incubate over night at room temperate;
33a) Centrifuge 60 min at 18,000 g and room temperate;
34a) Remove the isopropanol completely using a pipette;
35a) Re-suspend pellet in 50 µl diethyl pyrocarbonate (DEPC) treated
sterile dH2O.
a
In case extraction of RNA is desired, all used vessels must be free
of RNAse and solutions must be prepared with DEPC treated water.
2.2.4. Separation of DNA and RNA
The extracted nucleic acid solutions are subdivided into two equal
aliquots and treated with DNAse I and RNAse A (both Invitrogen),
respectively, as recommended by the manufacturer. Subsequently, the
respective nucleic acid fractions are precipitated as described in steps
31 to 34 of the protocol above, and re-suspended in 25 µl of DEPC
treated dH2O.
2.3. Additional extraction protocols tested
To evaluate the efficiency of the newly developed protocol,
corresponding subsamples were processed with the Fast DNA Spin
Kit for Soil (Q-Biogene) as well as according to the protocols of Griffiths
et al. (2000) and Hurt et al. (2001). Additionally, parallel subsamples
were washed applying the respective steps of the protocol of Fortin
et al. (2004) before crude nucleic acid extraction (steps 1 and 12 to 35
of the protocol above, with V5 = 650 µl), whereas the three washing
steps were repeated once to thrice. The crude extract of further
subsamples was purified by filtration through polyvinylpolypyrrolidone (PVPP) spin columns as described by Mendum et al. (1998).
2.4. Quality and quantity of nucleic acid extracts
The quality of the DNA extractions was controlled via spectrophotometry (NanoDrop, Peqlab). Contamination by co-extracted
humic substances was assessed via the absorbance ratio A260/230.
This ratio exceeds 2.0 for pure DNA. While ratios of 1.7 indicate nearly
pure DNA extractions from environmental samples (Bruns and
Buckley, 2002), we decided to use 1.5 as a threshold for reasonable
D. Peršoh et al. / Journal of Microbiological Methods 75 (2008) 19–24
quantification of nucleic acids using spectrophotometry, considering
the results of Bachoon et al. (2001). Likewise, the A260/280 ratio,
indicating contamination by proteins, is about 2.0 for pure DNA and
should exceed 1.7 for nearly pure nucleic acid extractions from
environmental samples. Finally, the A260/270 ratio (about 1.2 for pure
DNA) was measured to detect significant phenol contamination
(A260/270 b1.1), which would result in an overestimation of the nucleic
acid concentration (Stulnig and Amberger 1994). Of nucleic acid
extractions fitting the three criteria, concentrations were calculated
assuming an absorbance of 1.0 (10 mm path) at 260 nm, which
corresponds to a concentration of 46.7 ng/µl. The factor was deduced
from the results, which indicated a ratio of about 2:1 for DNA:RNA in
most samples and factors of 50 and 40 for pure DNA and RNA,
respectively.
3. Results
The relevant results of the preliminary tests are briefly summarized as follows. The bead beating instrument was used to thoroughly
mix the samples in step 5 of the protocol, because vortexing alone
proved not to be sufficient. However, two vortexing steps (45 s, max.
speed) with an intermediate centrifugation step (1 min at 3500 g)
provided similar results. Agarose gel electrophoresis revealed that,
while the tested extraction buffers yielded similar amounts of DNA,
RNA recovery was best using Tris–HCl buffer with LiCl, SDS and EDTA
(Kramer and Singleton, 1992). EtOH and isopropanol were similarly
efficient for the precipitation of nucleic acids (steps 31 and 32), both at
room temperature over night and at −20 °C for 2 h. However, the
volume of the precipitate obtained with isopropanol at room
temperature was less than 10% compared to the others, indicating a
low amount of co-precipitated salts.
A test series revealed that 1.5 µmol of Al2(SO4)3 suffice to efficiently
precipitate 1 mg of pure humic acid. The experiment also revealed that
15 min of mixing 1 mg humic acids added to 1 ml of water on a
vortexing device at maximum speed was insufficient to dissolve the
humic acids, while these were completely dissolved in 15 s using the
bead beating instrument.
According to the protocol for rough estimation of the required Al2
(SO4)3 concentration, 100 µmol of Al2(SO4)3 were needed to
precipitate all humic substances from the Of and Oh litter layers,
40 µmol for samples from the Ah, and 30 µmol for the Bv horizon. The
nucleic acid extractions revealed that addition of 80, 90, 40, and
40 µmol Al2(SO4)3 was necessary to obtain extracts with an A260/230
above 1.5 from the Of, Oh, Ah and Bv, respectively (Table 1). Addition of
excessive Al2(SO4)3 resulted in similar qualitative parameters, while
the total amount of extracted nucleic acids decreased.
The concentration of nucleic acids was highest in the uppermost
organic litter layer (Of) and decreased with soil depth, with the
highest difference between the upper litter layers and the lower soil
horizons. The DNA:RNA ratio within the extracts increased from 1.69
in Oh over 1.86 in Ah to 3.10 in Bv, while a median ratio of 2.12 was
found for the Of litter layer.
None of the additionally tested methods resulted in a nucleic acid
extract with A260/230 ratio above 1.5 (Fig. 1). The extracts obtained
according to the protocol of Hurt et al. (2001) and by the Fast DNA Spin
Kit for Soil (Q-Biogene) showed a maximum absorbance at about
230 nm (Fig. 2). The protocol by Griffiths et al. (2000) resulted in an
extract, the absorbance spectrum of which roughly matches that of
pure humic acids in water. The effect of soil washing (Fortin et al.,
2004) was negligible. Even after the washing steps had been
conducted thrice, the nucleic extracts were of nearly black color and
a spectrophotometric measurement was only possible for extracts
from the Bv horizon that contains the lowest concentration of humic
substances. The respective data are therefore not shown. Purification
of the crude extract with PVPP resulted in an absorbance spectrum
consistently decreasing from 220 to 350 nm. However, all measurable
21
extracts showed a minor bulge around 260 nm. While this was a bit
more distinct for extracts from the lower horizons that contain fewer
humic substances, the deviation from the trend line never exceeded
25% of the absorbance of the corresponding extract obtained with the
here optimized method.
4. Discussion
4.1. Extraction of nucleic acids from soil
The majority of the hitherto published protocols for the extraction
of nucleic acids from soil consist of two steps. First, nucleic acids are
extracted (“crude extract”) and subsequently co-extracted humic
substances are removed from the crude extract (Jackson et al., 1997;
Miller et al., 1999; Bruns and Buckley 2002; LaMontagne et al., 2002;
Luis et al., 2004; Luis et al., 2005; Arbeli and Fuentes 2007; Bernard
et al., 2007; Lakay et al., 2007; Weiss et al., 2007). However, humic
substances (i.e. humic acids) interact with all kinds of molecules
(Stevenson 1976), including nucleic acids, to which they may
covalently bind. Accordingly, the loss of nucleic acids during the
purification step is, with above 50%, immense (Howeler et al., 2003;
Carrigg et al., 2007). For this reason, it appears unlikely that the
recovered fraction sufficiently represents the nucleic acid spectrum
within the original soil sample. Interactions of the humic substances
with reagents applied during nucleic acid extraction may additionally
account for inconsistencies in analyses of microbial community with
different extraction techniques (LaMontagne et al., 2002; Carrigg et al.,
2007). Against this background, reliable comparative analyses of soils
with different humic substance composition appear virtually impossible. These problems may only be overcome by removal of humic
substances prior to cell lysis (Fortin et al., 2004; Dong et al., 2006).
While washing the soil samples (Fortin et al., 2004) proved
deficient with our samples, it may be feasible for samples with lower
humic substance content, or when repeated several times. In the latter
case, however, the procedure is very time-consuming and the stability
of the nucleic acids (mainly the RNA) may not be guaranteed. The aim
of the soil washing procedure, i.e. the removal of heavy metals and
Table 1
Absorbance ratios and nucleic acid concentrations of extracts from the two soil horizons
and the two litter layers, applying increasing Al2(SO4)3 concentrations
Layer /
horizon
AL2(SO4)3
added [µM]
A260/230
A260/280
A260/270
Nucleic acid yield
[µg] per gram soil
Wet
weight
Dry
weighta
Of
70
80
90
100
0.87
1.66
1.71
1.93
1.50
1.82
1.84
1.90
1.09
1.18
1.19
1.22
7b
28
25
20
21b
83
71
58
Oh
80
90
100
110
1.24
1.70
1.71
1.73
1.64
1.79
1.79
1.82
1.12
1.18
1.17
1.16
4b
18
14
10
12b
54
42
29
Ah
35
40
45
50
1.15
1.69
1.68
1.68
1.63
1.83
1.84
1.81
1.11
1.17
1.16
1.14
10b
13
13
9
14b
18
18
13
Bv
30
35
40
45
1.06
1.35
1.72
1.71
1.59
1.74
1.84
1.83
1.10
1.14
1.15
1.18
8b
8b
9
5
10b
11b
12
7
a
Water content analyzed of parallel samples by Kandeler et al. (sumitted for
publication).
b
value estimated by assuming the straight line through the absorbance values at
310 nm and 340 nm as baseline.
22
D. Peršoh et al. / Journal of Microbiological Methods 75 (2008) 19–24
Fig. 1. A260/230 ratio of nucleic acid extracts obtained using different extraction methods.
other contaminants of polluted environments, is also reached by the
quantitative elimination of humic substances, because these contaminants are readily bound by humic substances, wherefore they are
not removed from the nucleic acid extracts by standard isolation
protocols (Fortin et al., 2004).
While the original protocol using Al2(SO4)3 for flocculation of
humic substances (Dong et al., 2006) had minor weaknesses, the basic
principle proved very efficient. The here presented protocol, which is
based on this principle, resulted in nucleic acids extracts of high
purity. None of the extracts resulting from other tested protocols
matched the criteria for pure nucleic acids. Especially A260/230 ratios
below 1.5 revealed huge amounts of co-extracted humic substances
(Fig. 1). Due to the impurity of these extracts, there are no reliable data
to directly compare the quantity of nucleic acids extracted using these
and our protocols. Nevertheless, the absorbance spectra of the impure
extracts showed only a minor elevation of the absorbance at 260 nm
deviating from the expected absorbance spectrum of the humic
substances (Fig. 2). This deviation was always clearly below the
absorbance measured for the extracts obtained with the here
presented protocol. Hence, its application recovered the highest
concentration of nucleic acids.
4.2. Nucleic acid extraction protocol
A schematic overview on the presented extraction protocol is given
in Fig. 3, while its crucial steps are discussed in detail in the following.
Fig. 2. Absorbance spectra of nucleic acids extracts obtained applying different protocols, exemplified for samples of the litter layer Oh. The here presented protocol (Al-method) was
comparatively analyzed to purification of a crude extract using PVPP (both scaled on left y-axis), the Fast DNA Spin Kit for Soil (Q-Biogene), and the protocols of Griffiths et al. (2000)
and Hurt et al. (2001), the values for which refer to the right y-axis. The absorbance spectra of pure humic acids (Roth) in water (1 mg/ml, scaled on right y-axis) and pure nucleic
acids (0.1 mg/ml, DNA:RNA 2:1, scaled on left y-axis) are given for comparison.
D. Peršoh et al. / Journal of Microbiological Methods 75 (2008) 19–24
23
results reveal unambiguous trends. Due to the heterogeneous
structure of soil, the replicate number is insufficient to calculate
reasonable statistical support for the data. However, because the
purpose of this article was to make a powerful protocol available for
soil scientists and not to discuss the findings in a biological context, we
consider statistical analyses as being dispensable at this stage.
Therefore, representative data are presented throughout this article.
Nevertheless, a layout for reproducible quantitative studies is
proposed below.
4.4. Layout for quantitative studies
Fig. 3. Workflow for the extraction of nucleic acids from soil. For details see text.
A variable amount of Al2(SO4)3 may be added to the soil sample,
which allows the application of the protocol for a variety of soil types
with humic acid contents even higher than 200 mg per gram wet soil. At
step 5 of the protocol, the humic substances are flocculated by the Al3+
ions under acid conditions, as discussed in detail by Dong et al. (2006).
Vortexing alone was insufficient for a quantitative flocculation of the
humic substances, because the outer layers of humic substances
probably hamper detain the Al3+ ions to reach the inner layers. The
observation that mixing with the bead beating instrument is much more
effective in dissolving pure humic acids than using a vortexing device
additionally indicates that the bead beating instrument should be used
at step 5. At steps 6 to 9, the pH value is adjusted to 8 or above, to
precipitate excessive Al3+ as Al(OH)3. The finding that nucleic acid
recovery decreases with increasing Al2(SO4)3 concentrations (Table 1)
indicates that the precipitation of Al3+ ions is either not complete, or, that
formed Al(OH)+4 ions may precipitate nucleic acids as well. To minimize
the loss of nucleic acids and concurrently expand the range of Al2(SO4)3
concentrations suitable for maximum nucleic acid recovery, the
excessive solution is removed at step 11 of the protocol. Furthermore,
a beating step at low speed (step 16) was included to release humic
substances inaccessible for the Al3+ ions until then, while the majority of
cells present in the substrate remain intact at this step. Because of the
chemically similar nature of humic and nucleic acids, any substance in
solution capable of precipitating nucleic acids should precipitate with
the released humic acids. The composition of the beads (step 13) may be
adapted to particular needs, without affecting comparability of the
results. Steps 14 to 30 roughly follow standard nucleic acid extraction
protocols (Zhou et al., 1996; Jackson et al., 1997; Miller et al., 1999;
Griffiths et al., 2000; Hurt et al., 2001; Bruns and Buckley 2002), using a
previously published extraction buffer (Kramer and Singleton, 1992),
which proved most effective for simultaneous DNA and RNA recovery in
the preliminary tests. Protein bound humic substances, which may
cause a brown to black coloration of the supernatant at step 22, are
removed from the extracts at steps 23 to 25. Nucleic acids are
precipitated (step 31) with isopropanol at room temperature for
minimal co-precipitation of salt.
4.3. Reliability of data
The nucleic acid extractions according to each tested protocol have
been conducted at least thrice for each horizon and layer and the
By removal of humic substances prior to the nucleic acid isolation,
the presented protocol enables quantitative studies on nucleic acid
diversity (e.g., microarray analyses) and composition (e.g., DNA–RNA
proportion) for the first time. The fact, that the amount of extracted
nucleic acids increases until sufficient Al2(SO4)3 is added, confirms
that nucleic acids bind to the thereby removed humic substances and
indicates that a maximum of nucleic acids may be extracted with this
protocol. However, since soils are of heterogeneous structure, a
constant amount of sample net weight does not warrant constant
humic and nucleic acids contents. Especially, irregularities in the
colonization density of microorganisms due to the accumulation of
organic or inorganic material (e.g., decaying roots, mineral grains) and
corresponding variations in the amount of organic soil compounds
might lead to inconsistent results. Therefore, for obtaining reliable
results, sample series with different Al2(SO4)3 concentrations have to
be processed in parallel. Furthermore, parallels of these sample series
have to be analyzed. During nucleic acid extraction, the most errorprone step is certainly the quantitative removal of isopropanol from
the precipitate. The risk of inadvertent removal of nucleic acid
fractions increases with decreasing size of the precipitate. Accordingly, this risk is minimized by a maximum amount of nucleic acids in
the extract, which may be accomplished by pooling of parallel
samples.
For further analyses in the course of our ongoing project, we
designed the following sampling strategy. Six series of three samples
(18 samples in total) are processed for each soil sample. The three
samples of each series are treated with different Al2(SO4)3 concentrations, spanning the desired range. Subsequent to nucleic acid
extraction, all samples with humic substance (A260/230 b1.65), phenol
(A260/270 b1.15), or protein (A260/280 b1.75) contamination are discarded. From the remaining samples, the one with the highest nucleic
acid content is selected from each series. From the six remaining
samples those representing median nucleic acids contents are
selected and pooled, while those with clearly deviating contents are
discarded. If necessary (i.e. parallel analyses of DNA and RNA) the
obtained sample may be subsequently subdivided into parallel
subsamples for further processing.
Concluding, the here presented protocol allows for efficient
extraction of highly pure nucleic acids from soil. Because humic
substances are precipitated in a flexible step prior to cell lysis, it may
be used for various types of soil and related substrates, making
comparative results and quantitative analyses possible.
Acknowledgements
We thank Dirk Böttger (Göttingen) the for invaluable help during
the soil sampling. The excellent cooperation with our project partners
(working groups of Ellen Kandeler, Hohenheim and Georg Guggenberger, Halle) accounted for an efficient sample drawing. Thomas
Brune (Hohenheim) also readily shared his data on water content of
the soil samples with us. Andrea Kirpal (Bayreuth) is thanked for
assistance with the laboratory work. The project (RA 731/9-1 and BU
941/9-1) was funded by the Deutsche Forschungsgemeinschaft (DFG)
in a joined application (PAK 12).
24
D. Peršoh et al. / Journal of Microbiological Methods 75 (2008) 19–24
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