Extraction of DNA from soil

Patrick Robe
a,
*, Renaud Nalin
a
, Carmela Capellano
b
, Timothy M. Vogel
c
,
Pascal Simonet
c
a
LibraGen, 11, bd Einstein, 69100 Villeurbanne, France
b
Aventis Pharma, centre de recherche de Vitry-Alfortville, 13, quai Jules Guesde B. P. 14, 94403 Vitry sur Seine cedex, France
c
Center for Microbial Ecology, UMR CNRS 5557, University Claude Bernard LYON 1, 43, boulevard 11 Novembre 1918,
69622 Villeurbanne cedex, France
Received 21 March 2003; accepted 20 May 2003
Abstract
There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular
techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to
improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The rst approach
consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purication. The alternative
approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purication. Several
published methods describe the recovery of highly puried nucleic acids that are well-suited for molecular purposes even though
a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and
biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective.
2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved.
Keywords: Soils; DNA extraction; Direct lysis; Indirect extraction; Metagenome; Bacteria
1. Introduction
Prokaryotes are the most ubiquitous organisms on earth,
represented in all habitats, including soil, sediment, marine
and terrestrial subsurface, animals and plant tissues and play
a key role in the biogeochemical cycles of the biosphere and
represent an enormous reservoir of novel valuable molecules
for health or industry. The vast majority of enzymes and
antimicrobial products have been isolated from microorgan-
isms cultivated on articial media in the laboratory.
Soil appears to be a major reservoir of microbial genetic
diversity [11] and may be considered as a complex environ-
ment. This extreme complexity results from multiple inter-
acting parameters including soil texture and structure, water
content, pH, climatic variations and biotic activity. Soil tex-
ture and structure are mainly determined by sand, silt, clay
and organic matter content through the organization of
micro- and macro-aggregates. Microorganisms are heteroge-
neously distributed inside microaggregates and in macro-
porosities outside microaggregates [19,54]. Moreover, mi-
croorganisms strongly bind with soil particles (including
clay) through a variety of binding mechanisms [4] that re-
duce access to the whole bacteria.
Microbial ecology studies have provided a better under-
standing of (i) the structure of microbial communities and the
evolution of these communities under various climatic, biotic
and xenobiotic conditions and (ii) the activities of microor-
ganisms. For several years, investigations of microbial diver-
sity were mainly based on isolation and laboratory cultiva-
tion of bacteria which lead to underestimation of the true
diversity. Laboratory cultivation relied on a succession of
critical steps that increase the bias: (i) soil dispersion in order
to dislodge cells from inner soil compartments and from soil
surface particles; (ii) centrifugation-based separation of the
bacteria from soil and organic particles; (iii) plating of bac-
* Corresponding author.
E-mail address: pk.robe@libragen.com (P. Robe).
European Journal of Soil Biology 39 (2003) 183190
www.elsevier.com/locate/ejsobi
2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved.
doi:10.1016/S1164-5563(03)00033-5
terial suspension on various solid nutrient media; (iv) isola-
tion and characterization of individuals. Laboratory cultiva-
tion approaches remain of great interest when a selective
enrichment targets isolates for their ability to metabolize
specic substrates or to degrade deleterious xenobiotic com-
pounds. However, only bacterial isolates for which optimal
cultivation criteria have been determined can be recovered by
this approach.
The emerging use of molecular biology in microbial ecol-
ogy has revealed that less than 1% of the microorganisms
present in numerous environments are readily cultivable [2].
Molecular analysis of bacterial diversity in complex environ-
ments has relied on DNA/DNA hybridization, reassociation
kinetics studies [66] and mainly on 16S r DNAamplication,
(for review see Ref. [74]). However, these methods require
DNA extracts free of the numerous inhibitory contaminants
commonly found in environmental samples (for review see
Ref. [75]) and must exhibit an unbiased sampling of the
investigated bacterial community.
The aim of this review is to present an overview of soil
DNA extraction methods. Two approaches have been devel-
oped for extracting nucleic acids from soil samples. One is
the direct extraction of nucleic acids from soil samples after
in situ cell lysis which is then followed by DNA purication
[46]. The second approach separates the cell fraction from
soil particles rst. Then, this bacterial fraction is lysed and
the nucleic acids puried [10,23,67]. Both approaches have
advantages and disadvantages related to DNA yields, DNA
purity for molecular purposes, and the unbiased representa-
tion of the entire microbial diversity [10,58].
2. Direct lysis extraction method
The direct in situ lysis extraction method has been widely
used during the last decade. This method, which assumes
complete in situ lysis of all microorganisms, generally pro-
vides the highest DNA yields within acceptable processing
times.
All published techniques derived from the original proce-
dure of Ogram et al. [46] are summarized in two critical
steps. The disruption of the microbial cell wall is the rst step
leading to the release of all nucleic acids from all bacteria,
theoretically independent of the cell wall sensitivity to lysis,
the location of bacteria in microstructures, and their interac-
tions with soil particles. In the second step, nucleic acids are
separated from soil particles.
2.1. Cell lysis
Currently, three types of cell lysis (or membrane disrup-
tion) are used alone or in combination: (i) physical-, (ii)
chemical- and (iii) enzymatic-disruption.
Physical treatments, which destroy soil structure, tend to
have the greatest access to the whole bacterial community,
including bacteria deep within soil microaggregates. The
most commonly used physical disruption methods are freez-
ingthawing or freezingboiling [12,43,71] and bead-mill
homogenization [37,41,42,63]. When testing a bead beat-
ing method, Brgmann et al. [7] established that DNA
yields increased with longer beating times, higher speed and
reduced extraction buffer volumes but at the cost of DNA
shearing.
Other methods, which use mortar mill grinding [64],
grinding under liquid nitrogen [73,77], ultrasonication
[48,51] and microwave thermal shock [47] have also been
reported. Frostegard et al. [17] observed that drying soil
before grinding greatly improved the lysis efciency.
In general, physical methods have shown efciencies for
disruption of vegetative forms, small cells and spores [43]
but they often result in signicant DNA shearing [35,62].
Chemical lysis either alone or in association with physical
methods have been used extensively. Probably the most com-
mon chemical is the detergent sodium dodecyl sulfate (SDS)
which dissolves the hydrophobic material of cell mem-
branes. Detergents have often been used in combination with
heat-treatment and with chelating agents such as EDTA,
Chelex 100 [21,28] and diverse Tris buffer or sodium phos-
phate buffers [29]. In several studies, increasing the EDTA
concentration increased the strength of extraction and lysis
buffers resulting in higher yields, but lower purity of isolated
nucleic acids. This result leads to the conclusion that the
choice of buffer is a compromise between the expected DNA
quantity and the required DNA purity. In a comparative
study, Miller et al. [42] reported that the Chelex 100 treat-
ment had detrimental effect by decreasing the DNA yield. A
rapid protocol was reported by Selenska and Klingmller
[60] based only on gentle shaking of soil slurry in a SDS
sodium phosphate buffer at 70 C. Nucleic acids recovered
from this gentle procedure were on average approximately
25 kbp.
Addition of cetyltrimethyl-ammonium bromide (CTAB)
and polyvinylpolypyrrolidone (PVPP) has also been reported
[29,44,77]. Both CTAB and PVPP can partially remove hu-
mic compounds, but (contrary to CTAB) PVPP resulted in
DNAloss [77]. CTABforms insoluble complexes with dena-
tured proteins, polysaccharides and cell debris [59]. PVPP
was shown to be ineffective during cell lysis, but efcient
when used as a spin column during the nucleic acids puri-
cation step [29].
Another denaturing agent, guanidine isothiocyanate has
been used for mRNAextraction fromseeded soils [47,71] but
has [71] not proven valuable when extracting DNA from soil
samples [37,42].
Many protocols including an enzymatic lysis have been
developed. Lysozyme treatment is one of the most common
[6,37,55,64,70]. Improvement in DNA purity may result
from the lysozyme hydrolytic action on glycosidic or other
humic components bonds. Another enzyme, achromopepti-
dase, was used to improve the lysis of the recalcitrant Gram
positive Frankia [61] and another, proteinase K, was used to
digest contaminating proteins [37,50,77].
184 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
2.2. Nucleic acid extraction and purication
Several methods for separating and purifying nucleic ac-
ids from soil components have been investigated. A major
soil component, humic acid, inhibits restriction enzyme di-
gestion of DNA, the polymerase chain reaction [64] and also
alters the results of quantitative membrane hybridizations by
lowering the expected hybridization signal [1]. The phenolic
groups in humic acids denature biological molecules by
bonding to amides or are oxidized to form a quinone which
covalently bonds to DNA [76]. In most studies, following
cell lysis DNA a rst purication step of DNA is achieved by
organic solvent extraction (either phenol or chloroform),
followed by ethanol, isopropanol or polyethylene glycol-
assisted precipitation [63]. These crude extracts have been
treated by numerous methods based on DNA physical and
chemical properties. Nucleic acid binding on hydroxyapatite
columns was successful for extracting DNA[46,63] and both
DNA and rRNA [52] from soil and sediment samples. Ce-
sium chloride (CsCl) density gradient centrifugation was
also shown to allow further enzymatic restriction of puried
nucleic acids [46,50]. In contrast, Steffan et al. [63] noted
that even extensive purication with CsCl gradient centrifu-
gation and hydroxyapatite chromatography resulted in DNA
loss and did not remove all humic acids. Since heterogeneous
humic acids were dispersed throughout the CsCl gradient,
some humic contaminants were coextracted with the DNA
band, requiring a second CsCl gradient centrifugation to
dilute these residual humic acids. Other purication proto-
cols that are less time-consuming and adaptable to the pro-
cessing of a higher number of samples have been used indi-
vidually or in combination [18]:
1. Agarose gel electrophoresis [18,43,56,77]. DNA frag-
ments may be recovered from gel using electroelution
[34]. Gel electrophoresis assisted by polyvinylpyrroli-
done (PVP) was reported to facilitate separation of
DNA from humic acids [24,31] since PVP retards the
phenolic compounds that usually co-migrate with
nucleic acids [32,76].
2. Gel ltration resins including sephadex G200 [30] and
G150, Sepharose 2B, 4B and 6B, Biogel P100 and
P200 have been evaluated in comparative studies
[26,37,41]. Sepharose resins demonstrated a better
separation of DNA from humic acids. Microspin
Sephacryl S-300 and S-400 columns (Pharmacia Bio-
tech) were also used to purify crude DNA extracts
contaminated with humic acids [15,17].
3. Other commercial purication products were also in-
vestigated such as Wizard DNA clean-up system
(Promega) [20] and Centricon 50 and Microcon
100 concentrators (Amicon) [77], Elutip D column
from Schleicher & Schuell [12,17], silica-based DNA
binding SpinBind Columns from FMC BioProducts
[42] and Tip-100 and Tip-500 columns from Qiagen
[25,64], respectively. Tebbe and Vahjen [64] showed
that 97% of the initially coextracted humic acids could
be removed through ion-exchange chromatography.
Ecological and molecular PCR-based studies require ex-
tensive purication of nucleic acids extracts in order to re-
move humic acids and other contaminants. DNA losses dur-
ing extensive purication may compromise the detection of
rare DNA sequences. Jacobsen [27] successfully removed
the PCR-inhibitory effect of humic acids through an alterna-
tive magnetic capture hybridization (MCH) approach. MCH
separates a specic DNA target from all other DNA and
contaminants, including humic acids. A specic single-
stranded DNA, labelled with biotin, is used to hybridize the
unpuried nucleic acids of soil sample. After recovery of
specic DNADNA hybrids by a magnetic extraction and
washing, PCR amplication can be performed. Chandler et
al. [8] similarly investigated afnity magnetic capture using
peptide nucleic acids (PNA) clamps and oligomers as spe-
cic hybridization probes. These afnity capture approaches
are, therefore, particularly promising when specic DNA
sequences are targeted in very small soil samples or in low
biomass soil samples.
2.3. Toward a universal lysis extraction method?
Although numerous direct lysis extraction protocols re-
sulting from complex combinations of physical, chemical,
and/or enzymatic methods to purify nucleic acids have been
published application has been limited to only a fewsamples.
Therefore, the individual contribution of each step to the nal
success of DNA recovery cannot be clearly evaluated. Even
when evaluation is attempted, the positive or negative effects
of extraction parameters is difcult to extrapolate to other
samples. However, some comparative studies have contrib-
uted to better understanding the value of extraction and/or
purication steps when applied to previously characterized
soil samples [10,17,25,33,42,77].
Unbiased nucleic acid-based access to bacterial diversity
in environmental samples requires several conditions:
1. All bacteria must be released from soil compartments.
This includes those bacteria which are strongly ad-
sorbed on soil colloids or those located within the inner
microporosity of soil aggregates [54]. Hattori [19] ob-
served that culturable Gram-positive bacteria were
more abundant in soil macropores whereas Gram-
negative bacteria predominated in micropores. Physi-
cal disruption methods such as bead-mill homogeniza-
tion and freezingthawing helped homogenize soil
rendering conned bacteria available for lysis treat-
ments [17], and thus lead to better DNA yields.
2. Lysis of recalcitrant organisms such as Gram-positive
cells, spores and small bacteria requires harsh treat-
ments that may result in DNA shearing of lysis-
sensitive bacteria.
3. Nucleic acid extraction should be performed as soon as
possible after sample collection since storing samples
at 4 C for several weeks can result in the degradation
of the large molecular-weight DNA fraction [65].
185 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
Although recovery of large DNA fragments (4090 kb)
using gentle lysozymeSDS-based methods was reported
[29], satisfactory DNA yields and unbiased DNA extraction
require the introduction of mechanical lysis treatments
which commonly result in DNA shearing. In most studies,
direct extraction did not lead to the recovery of DNA frag-
ments larger than 20 kb. Some applications, such as construc-
tion of metagenomic libraries, require the extraction of large
DNA fragments that are rarely obtained by direct extraction
methods.
3. Bacteria extraction method
Bacterial extraction was rst reported by Faegri et al. [16]
and Torsvik and Goksoyr [69] and is based on the following
sequential steps: (i) dispersion of soil particles, (ii) separa-
tion of the cells from soil particles by centrifugation accord-
ing to sedimentation velocities, buoyant density or both, (iii)
lysis of extracted cells and (iv) DNA purication [4].
3.1. Soil dispersion methods
As for the direct lysis extraction approach, dispersion of
the soil sample by physical and/or chemical methods is a
critical step because of the location of microorganisms
within soil aggregates and because of bacterial adhesion to
soil particles. Waring blender dispersion has been widely
used [4,16]. Other methods have been tested, such as sonica-
tion [53], mild dispersal by shaking [72], and rotating pestle
[36]. When compared, waring blender and rotating pestle
proved to be the most efcient dispersion methods for large
and small soil sample volumes, respectively [36].
Chemical dispersal has often been used in combination
with mechanical methods [36]. Cation exchange resins
(Chelex 100) have proven efcient for soil dispersion [28].
Bakken [3] did not nd a signicant dispersion difference
between hexametaphosphate and distilled water. Many other
chemical agents have been evaluated: specic detergents
(sodium cholate and sodium deoxycholate), which interact
with bacterial lipopolysaccharides [40], polyethylene glycol
(PEG) and SDS which dissolve hydrophobic material [63],
PVPP which removes humic acids [63]. However, chemical
agents may have adverse side effects. For example, the meta-
bolic state of soil bacteria as measured by cellular adenosine
triphosphate levels was negatively affected when a combined
SDSChelex chemical treatment was applied [4]. Preserva-
tion of bacterial integrity during cell separation seems to be
essential in order to prevent released DNA to be degraded by
physical, chemical and/or enzymatic processes.
3.2. Centrifugation-based cell separation
Separation of bacteria from soil particles according to
sedimentation velocities was rst described by Faegri et al.
[16]. This method is based on two successive centrifugations:
rst a low speed centrifugation ranging from 500 g to
1000 g lasting 215 min, respectively, in order to remove
(sediment) soil debris, fungal mycelia and heavy soil par-
ticles [16]. Optimal conditions for low-speed centrifugation
is a compromise between cell recovery efciency and elimi-
nation of contaminating material [63]. A subsequent high-
speed centrifugation of the cell-containing supernatant pro-
duces the bacterial fraction. Chains and clusters of bacteria
and bacteria attached to soil particles might be easily lost
during the rst centrifugation. Holben et al. [23] established
that approximately 10% of the total bacteria present in the
soil were released per round of homogenization-separation.
According to these authors, the bacterial fraction released
after a single round is apparently as representative of the total
bacterial population as after multiple rounds. Several rounds
of extraction may, however, greatly improve total cell recov-
ery.
Supernatants resulting from the low speed centrifugation
might still contain non-cellular material and soil contami-
nants such as humic acids. Flocculation of the cell debris and
clay particles using CaCl
2
resulted in a considerable decrease
in cell recovery [28]. An alternative high-speed centrifuga-
tion method based on density gradient centrifugation, was
developed [3] in order to separate bacteria according to their
buoyant density. Several multi-gradient media have been
tested such as Percoll, metrizamide and Nycodenz [4] but the
use of Nycodenz provided the best results. The dispersed soil
suspension is deposited onto Nycodenz cushion
(p = 1.3 g ml
1
) and then centrifuged at high speed, com-
monly 10,000 g. Bacteria will settle on top of the Nycodenz
cushion, and organic and mineral particles of greater den-
sity will sediment to the bottom of the tube. Extraction
efciency (number of extracted bacteria as a percentage of
total bacteria in the soil suspension), was between 6% and
25% [39] and from 20% to 50% [4]. The organic matter and
clay content of the soil will affect the cell extraction ef-
ciency [36] conrming that considerable attention needs to
be paid to soil dispersion procedures (as described above).
Even if most bacteria have densities below 1.12 g ml
1
and
should be recovered on the Nycodenz cushion, many bacteria
are strongly attached to dense soils particles, such as clays
and will sediment through the gradient. Although the bacte-
rial fraction will still contain humic material and soil par-
ticles having the same density as the bacteria [68], centrifu-
gation on Nycodenz gradient allows recovery of a relatively
clean bacterial fraction as compared to low speed centrifuga-
tion methods. A similar method described by Pillai et al. [49]
was based on sucrose density gradient centrifugation.
3.3. Nucleic acid separation and purication
Bacterial DNA can be extracted from the puried cellular
fraction using numerous physical, chemical and enzymatic
procedures as mentioned above. Cesium chlorideEthidium
bromide equilibrium density centrifugation has been suc-
cessfully used to recover pure DNA [28,65] of large size (at
least 48 kb) [9,23].
186 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
Torsvik [67] developed a protocol in which the bacterial
lysate was puried by passage through a hydroxyapatite
column. A high urea concentration was used to disrupt hy-
drogen bonds between DNA and humic acids. Steffan et al.
[63] observed that the removal of humic acids by PVPP
greatly improved the DNA purity but lowered the DNA
recovery slightly. CsCl density centrifugation and hydroxya-
patite column chromatographic purication improved the
DNA purity, as measured by the spectrophotometric A
260/280
and A
260/230
ratio, but both caused the loss of DNA [63].
Gentle lysis methods combined with CsCl density puri-
cation recovered highly puried nucleic acids with improved
sizes reaching more than 100 kb (Robe, unpublished data).
The DNA size remains limitative for functional genomic
approaches where the exploration of gene clusters and bio-
synthetic pathways through cosmid and bacterial articial
chromosome (BAC) cloning requires DNA greater than
200 kb.
Embedding bacteria in agarose plugs before performing a
gentle bacterial lysis recovered DNA fragments of a few
hundred kilobases with limited mechanical shearing. This
approach was used to establish libraries of BAC clones con-
taining large DNA fragments from several microorganisms
including Methanosarcina thermophila [14], Bacillus cereus
[57], Mycobacterium tuberculosis [5] and Pseudomonas
aeruginosa [13]. An integrated approach combining centri-
fugation-based cell separation from soil particles, in plug
lysis and pulsed eld gel electrophoresis (PFGE) has been
successfully applied to non-culturable bacteria fromenviron-
mental samples [45]. DNA fragments recovered using this
method are more than 300 kbp in size and of adequate purity
for further molecular cloning procedures [45]. This method
has already lead to the construction of a soil fosmid metage-
nomic library containing more than 3.5 Gbp providing access
to metagenomic functional genetics (Nalin and Robe, unpub-
lished results).
4. Comparative evaluation of direct and indirect
methods
In a comparative study between several extraction meth-
ods, Leff et al. [33] established that the bead-beating-SDS
method of Ogram et al. [46] allowed the greatest DNA yield
but resulted in increased DNA shearing when compared to
the comparatively gentle freezing-thawing lysozyme method
described by Tsai and Olson [70]. Compared to these two
direct extraction methods, the indirect extraction method of
Jacobsen and Rasmussen [28] based on centrifugation recov-
ery of cell fraction before lysis and CsCl DNA purication,
gave the lowest DNA yield but showed the highest DNA
purity with a low degree of DNA shearing. Tien et al. [65]
reported that the direct lysis methods produced similarly
DNA yield at least a 10-fold greater than the indirect extrac-
tion methods.
The main disadvantage of cell fractionation-based meth-
ods is that the recovered bacterial fraction represents only
2550% of the total endogenous bacterial community [4]. In
return direct extraction lysis has been assumed to recover
more than 60% of the total theoretical bacterial DNA [43].
Miller et al. [42] reported DNA recovery efciencies ranging
from 86% to more than 100%, based on direct counts of
endogenous microorganisms [42]. These values probably
overestimate the recovery efciencies, resulting from the
eukaryotic and extracellular DNA recovery and the underes-
timation of bacteria by uorescent direct counts.
Even though indirect extraction methods typically result
in lower humic acid contamination than direct extraction
methods, both approaches rely on subsequent purication
steps in order to produce nucleic acids usable for molecular
purposes. When comparing both direct and indirect methods,
Steffan et al. [63] established that the degree of purity re-
quired for restriction endonuclease digestion of recovered
DNA varied with the enzyme used. While PvuII did not
require further hydroxyapatite purication after CsCl puri-
cation, target DNA restriction by EcoRI was only observed
with DNA recovered by cell fractionation method using the
most extensive CsCl-hydroxyapatite purication. Endonu-
clease restriction by SalI failed for both extraction methods.
Clearly, comprehensive knowledge of the sensitivity of mo-
lecular methods to inhibitory effect of contaminants coex-
tracted with environmental DNA is a prerequisite when es-
tablishing an extraction and purication strategy.
Denaturing gradient gel electrophoresis (DGGE) was
used by several authors to detect differences between extrac-
tion methods [22,29,37]. The DGGE band pattern was af-
fected by the extraction procedure as indicated by the varia-
tion of band intensity [29]. Such sources of bias were also
observed by Maarit Niemi et al. [37] when comparing DGGE
community proles in rhizosphere soils samples.
Ribosomal intergenic spacer analysis (RISA) of soil mi-
crobial diversity also revealed that both abundance and ap-
parent members of the bacterial community are affected by
the extraction method [38]. By using hybridization of the
PCR amplied 16S rDNA gene Courtois et al. [10] did not
nd signicant difference in the spectrum of diversity result-
ing from the two extraction strategies, the only exception
being the gamma-subclass of Proteobacteria.
5. Conclusion
Both direct and indirect extraction methods can recover
puried nucleic acids for molecular biology purposes. Direct
extraction methods have been developed for several reasons;
they are assumed to provide a less biased extraction of
microbial DNA, with less laborious methods and higher
nucleic acid yields. Direct lysis procedures are preferred
when large quantities of nucleic acids are required for DNA-
consuming methods, statistically signicant detection of
non-abundant microorganisms, and when the entire diversity
of an environmental sample must be investigated with mini-
mum bias. However, the resulting nucleic acid extracts are
commonly sheared and contaminated with humic acids. Fur-
187 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
thermore, extracts often contain unknown amounts of extra-
cellular and/or eukaryotic DNA.
Although time-consuming, indirect extraction methods
are preferred for targeting prokaryotic communities, when
high DNA purity is required for inhibitor-sensitive methods,
and when recovery of high molecular weight DNA is neces-
sary.
For both direct and indirect extraction methods, several
parameters may be varied to optimize soil dispersion, cell
disruption, and separation and purication of nucleic acids.
Evidently, there is no single ideal extraction method and the
denition of an optimal strategy will rely on several ele-
ments:
1. A comprehensive description of soil is necessary.
2. Is co-extraction of extracellular and eukaryotic nucleic
acids acceptable and to which limit?
3. Targeted organisms need to be dened (the whole com-
munity or a specic taxon or group of taxons).
4. Whether the targeted microorganisms can be easily
recovered or whether they are conned inside inner
sample aggregates or strongly adsorbed on particle
surfaces needs to be considered.
5. The targeted microorganisms sensitivity to lysis proce-
dures needs to be determined.
6. Constraints caused by non-satisfactory DNA quantity
and purity will greatly inuence the choice of extrac-
tion and purication methods, as previously discussed.
Undoubtedly, an unbiased description of microbial diver-
sity depends on the optimization of nucleic acid extraction
methods. In addition, considerable attention will need to be
paid to other major sources of bias introduced by environ-
mental sampling protocols and molecular biology methods.
Acknowledgements
This work was nancially supported by the Euro-
pean project TRANSBACNo. QLK3-CT-2001-02242.
References
[1] E.W. Alm, D. Zheng, L. Raskin, The presence of humic sub-
stances and DNA in RNA extracts affects hybridization
results, Appl. Environ. Microbiol. 66 (2000) 45474554.
[2] R.I. Amann, W. Ludwig, K.H. Schleifer, Phylogenetic identi-
cation and in situ detection of individual microbial cells
without cultivation, Microbiol. Rev. 59 (1995) 143169.
[3] L.R. Bakken, Separation and purication of bacteria fromsoil,
Appl. Environ. Microbiol. 49 (1985) 14821487.
[4] L.R. Bakken, V. Lindahl, Recovery of bacterial cells fromsoil,
in: J.D. Van Elsas, J.T. Trevors (Eds.), Nucleic Acids in the
Environment: Methods and Applications, Springer-Verlag,
Heidelberg, Germany, 1995, pp. 927.
[5] R. Brosch, S.V. Gordon, A. Billault, T. Garnier, K. Eiglmeier,
C. Soravito, B.G. Barrell, S.T. Cole, Use of a Mycobacterium
tuberculosis H37Rv bacterial articial chromosome library
for genome mapping, sequencing, and comparative genomics,
Infect. Immun. 66 (1998) 22212229.
[6] K.D. Bruce, W.D. Hiorns, J.L. Hobman, A.M. Osborn,
P. Strike, D.A. Ritchie, Amplication of DNA from native
populations of soil bacteria by using the polymerase chain
reaction, Appl. Environ. Microbiol. 58 (1992) 34133416.
[7] H. Brgmann, M. Pesaro, F. Widmer, J. Zeyer, A strategy for
optimizing quality and quantity of DNA extracted from soil,
J. Microbiol. Methods 45 (2001) 720.
[8] D.P. Chandler, J.R. Stults, S. Cebula, B.L. Schuck,
D.W. Weaver, K.K. Anderson, M. Egholm, F.J. Brockman,
Afnity purication of DNA and RNA from environmental
samples with peptide nucleic acid clamps, Appl. Environ.
Microbiol. 66 (2000) 34383445.
[9] S. Courtois, C.M. Cappellano, M. Ball, F.X. Francou, P. Nor-
mand, G. Helynck, A. Martinez, S.J. Kolvek, J. Hopke,
M.S. Osburne, P.R. August, R. Nalin, M. Guerineau, P. Jean-
nin, P. Simonet, J.L. Pernodet, Recombinant environmental
libraries provide access to microbial diversity for drug discov-
ery from natural products, Appl. Environ. Microbiol. 69
(2003) 4955.
[10] S. Courtois, A. Frostegard, P. Goransson, G. Depret, P. Jean-
nin, P. Simonet, Quantication of bacterial subgroups in soil:
comparison of DNA extracted directly from soil or from cells
previously released by density gradient centrifugation, Envi-
ron. Microbiol. 3 (2001) 431439.
[11] T.P. Curtis, W.T. Sloan, J.W. Scannell, Estimating prokaryotic
diversity and its limits, Proc. Natl. Acad. Sci. USA 99 (2002)
1049410499.
[12] V. Degrange, R. Bardin, Detection and counting of nitrobacter
populations in soil by PCR, Appl. Environ. Microbiol. 61
(1995) 20932098.
[13] K. Dewar, L. Sabbagh, G. Cardinal, F. Veilleux, F. Sanscha-
grin, B. Birren, R.C. Levesque, Pseudomonas aeruginosa
PAO1 bacterial articial chromosomes: strategies for map-
ping, screening, and sequencing 100 kb loci of the 5.9 Mb
genome, Microb. Comp. Genomics 3 (1998) 105117.
[14] S.V. Diaz-Perez, F. Alatriste-Mondragon, R. Hernandez,
B. Birren, R.P. Gunsalus, Bacterial articial chromosome
(BAC) library as a tool for physical mapping of the archaeon
Methanosarcina thermophila TM-1, Microb. Comp. Genom-
ics 2 (1997) 275286.
[15] V.P. Edgcomb, J.H. McDonald, R. Devereux, D.W. Smith,
Estimation of bacterial cell numbers in humic acid-rich salt
marsh sediments with probes directed to 16S ribosomal DNA,
Appl. Environ. Microbiol. 65 (1999) 15161523.
[16] A. Faegri, V.L. Torsvik, J. Goksoyr, Bacterial and fungal
activities in soil: separation of bacteria and fungi by a rapid
fractionated centrifugation technique, Soil Biol. Biochem. 9
(1977) 105112.
[17] A. Frostegard, S. Courtois, V. Ramisse, S. Clerc, D. Bernillon,
F. Le Gall, P. Jeannin, X. Nesme, P. Simonet, Quantication of
bias related to the extraction of DNAdirectly fromsoils, Appl.
Environ. Microbiol. 65 (1999) 54095420.
[18] M. Harry, B. Gambier, E. Garnier Sillam, Soil conservation for
DNA preservation for bacterial molecular studies, Eur. J. Soil
Biol. 36 (2000) 5155.
[19] T. Hattori, Soil aggregates as microhabitats of microorgan-
isms, Biol. Fertil. Soils 6 (1988) 189203.
188 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
[20] A. Henne, R. Daniel, R.A. Schmitz, G. Gottschalk, Construc-
tion of environmental DNA libraries in Escherichia coli and
screening for the presence of genes conferring utilization of
4-hydroxybutyrate, Appl. Environ. Microbiol. 65 (1999)
39013907.
[21] P.R. Herron, E.M.H. Wellington, New method for extraction
of Streptomycete spores from soil and application to the study
of lysogeny in sterile amended and non sterile soil, Appl.
Environ. Microbiol. 56 (1990) 14061412.
[22] H. Heuer, M. Krsek, P. Baker, K. Smalla, E.M. Wellington,
Analysis of actinomycete communities by specic amplica-
tion of genes encoding 16S rRNA and gel-electrophoretic
separation in denaturing gradients, Appl. Environ. Microbiol.
63 (1997) 32333241.
[23] W.E. Holben, J.K. Jansson, B.K. Chelm, J.M. Tiedje, DNA
probe method for the detection of specic microorganisms in
the soil bacterial community, Appl. Environ. Microbiol. 54
(1988) 703711.
[24] P. Hugenholtz, B.M. Goebel, N.R. Pace, Impact of culture-
independent studies on the emerging phylogenetic view of
bacterial diversity, J. Bacteriol. 180 (1998) 47654774.
[25] R.A. Hurt, X. Qiu, L. Wu, Y. Roh, A.V. Palumbo, J.M. Tiedje,
J. Zhou, Simultaneous recovery of RNA and DNA from soils
and sediments, Appl. Environ. Microbiol. 67 (2001)
44954503.
[26] C.R. Jackson, J.P. Harper, D. Willoughby, E.E. Roden,
P.F. Churchill, A simple efcient method for the separation of
humic substances and DNA from environmental samples,
Appl. Environ. Microbiol. 63 (1997) 49934995.
[27] C.S. Jacobsen, Microscale detection of specic bacterial DNA
in soil with a magnetic capture-hybridization and PCR ampli-
cation assay, Appl. Environ. Microbiol. 61 (1995)
33473352.
[28] C.S. Jacobsen, O.F. Rasmussen, Development and application
of a new method to extract bacterial DNA from soil based on
separation of bacteria from soil with cation-exchange resin,
Appl. Environ. Microbiol. 58 (1992) 24582462.
[29] M. Krsek, E.M. Wellington, Comparison of different methods
for the isolation and purication of total community DNA
from soil (In Process Citation), J. Microbiol. Methods 39
(1999) 116.
[30] C.R. Kuske, K.L. Banton, D.L. Adorada, P.C. Stark, K.K. Hill,
P.J. Jackson, Small-scale DNA sample preparation method for
eld PCR detection of microbial cells and spores in soil, Appl.
Environ. Microbiol. 64 (1998) 24632472.
[31] M.G. Lamontagne, F.C. Michel, P.A. Holden, C.A. Reddy,
Evaluation of extraction and purication methods for obtain-
ing PCR-ampliable DNA from compost for microbial com-
munity analysis, J. Microbiol. Methods 49 (2002) 255264.
[32] S.Y. Lee, J. Bollinger, D. Bezdicek, A. Ogram, Estimation of
the abundance of an uncultured soil bacterial strain by a
competitive quantitative PCR method, Appl. Environ. Micro-
biol. 62 (1996) 37873793.
[33] L.G. Leff, J.R. Dana, J.V. McArthur, L.J. Shimkets, Compari-
son of methods of DNA extraction from stream sediments,
Appl. Environ. Microbiol. 61 (1995) 11411143.
[34] W. Liesack, E. Stackebrandt, Occurrence of novel groups of
the domain bacteria as revealed by analysis of genetic material
isolated from an Australian terrestrial environment, J. Bacte-
riol. 174 (1992) 50725078.
[35] W. Liesack, H. Weyland, E. Stackebrandt, Potential risks of
gene amplication by PCRas determined by 16S rDNAanaly-
sis of a mixed-culture of strict barophilic bacteria, Microb.
Ecol. 21 (1991) 191198.
[36] V. Lindahl, L.R. Bakken, Evaluation of methods for extraction
of bacterial from soil, FEMS Microbiol. Ecol. 16 (1995)
135142.
[37] R. Maarit Niemi, I. Heiskanen, K. Wallenius, K. Lindstrom,
Extraction and purication of DNA in rhizosphere soil
samples for PCR-DGGE analysis of bacterial consortia,
J. Microbiol. Methods 45 (2001) 155165.
[38] F. Martin-Laurent, L. Philippot, S. Hallet, R. Chaussod,
J.C. Germon, G. Soulas, G. Catroux, DNA extraction from
soils: old bias for new microbial diversity analysis methods,
Appl. Environ. Microbiol. 67 (2001) 23542359.
[39] C. Mayr, A. Winding, N.B. Hendriksen, Community level
physiological prole of soil bacteria unaffected by extraction
method, J. Microbiol. Methods 36 (1999) 2933.
[40] R.M. McDonald, Sampling soil microoras: dispersion of soil
by ion exchange and extraction of specic microorganims
from suspension by elutriation, Soil Biol. Biochem. 18 (1986)
399406.
[41] D.N. Miller, Evaluation of gel ltration resins for the removal
of PCR-inhibitory substances from soils and sediments,
J. Microbiol. Methods 44 (2001) 4958.
[42] D.N. Miller, J.E. Bryant, E.L. Madsen, W.C. Ghiorse, Evalu-
ation and optimization of DNA extraction and purication
procedures for soil and sediment samples, Appl. Environ.
Microbiol. 65 (1999) 47154724.
[43] M.I. More, J.B. Herrick, M.C. Silva, W.C. Ghiorse, E.L. Mad-
sen, Quantitative cell lysis of indigenous microorganisms and
rapid extraction of microbial DNAfromsediment, Appl. Envi-
ron. Microbiol. 60 (1994) 15721580.
[44] R. Nalin, P. Normand, P. Simonet, A.M. Domenach, Poly-
merase chain reaction and hybridization on DNA extracted
from soil as a tool for Frankia spp. population distribution
studies in soil, Can. J. Bot. 77 (1999) 12391247.
[45] R. Nalin, R.P. Robe, V. Tran Van, Method for indirectly
extracting non-cultivable DNA organisms and DNA by said
method, France, Patent 01/11/2001, 2001.
[46] A. Ogram, G.S. Sayler, T. Barkay, The extraction and puri-
cation of microbial DNA from sediments, J. Microbiol. Meth-
ods 7 (1987) 5766.
[47] M. Orsini, V. Romano-Spica, A microwave-based method for
nucleic acid isolation fromenvironmental samples, Lett. Appl.
Microbiol. 33 (2001) 1720.
[48] C. Picard, C. Ponsonnet, E. Paget, X. Nesme, P. Simonet,
Detection and enumeration of bacteria in soil by direct DNA
extraction and polymerase chain reaction, Appl. Environ.
Microbiol. 58 (1992) 27172722.
[49] S.D. Pillai, K.L. Josephson, R.L. Bailey, C.P. Gerba, I.L. Pep-
per, Rapid method for processing soil samples for polymerase
chain reaction amplication of specic gene sequences, Appl.
Environ. Microbiol. 57 (1991) 22832286.
[50] L.A. Porteous, J.L. Armstrong, Recovery of bulk DNA from
soil by a rapid, small-scale extraction method, Curr. Micro-
biol. 22 (1991) 345348.
[51] L.A. Porteous, R.J. Seidler, L.S. Watrud, An improved method
for purifying DNA from soil for polymerase chain reaction
amplication and molecular ecology applications, Mol. Ecol.
6 (1997) 787791.
189 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190
[52] K.J. Purdy, T.M. Embley, S. Takii, D.B. Nedwell, Rapid
extraction of DNA and rRNA from sediments by a novel
hydroxyapatite spin-column method, Appl. Environ. Micro-
biol. 62 (1996) 39053907.
[53] A.J. Ramsay, Extraction of bacteria from soil: efciency of
shaking or ultrasonication as indicated by direct counts and
autoradiography, Soil Biol. Biochem. 16 (1984) 457481.
[54] L. Ranjard, F. Poly, J. Combrisson, A. Richaume, S. Nazaret,
A single procedure to recover DNA from the surface or inside
aggregates and in various size fractions of soil suitable for
PCR-based assays of bacterial communities, Eur. J. Soil Biol.
34 (1998) 8997.
[55] P.A. Rochelle, J.C. Fry, R.J. Parkes, A.J. Weightman, DNA
extraction for 16S rRNA gene analysis to determine genetic
diversity in deep sediment communities, Fems. Microbiol.
Lett. 79 (1992) 5965.
[56] M.R. Rondon, P.R. August, A.D. Bettermann, S.F. Brady,
T.H. Grossman, M.R. Liles, K.A. Loiacono, B.A. Lynch,
I.A. MacNeil, C. Minor, C.L. Tiong, M. Gilman,
M.S. Osburne, J. Clardy, J. Handelsman, R.M. Goodman,
Cloning the soil metagenome: a strategy for accessing the
genetic and functional diversity of uncultured microorgan-
isms, Appl. Environ. Microbiol. 66 (2000) 25412547.
[57] M.R. Rondon, S.J. Raffel, R.M. Goodman, J. Handelsman,
Toward functional genomics in bacteria: analysis of gene
expression in Escherichia coli from a bacterial articial chro-
mosome library of Bacillus cereus, Proc. Natl. Acad. Sci. USA
96 (1999) 64516455.
[58] C.L. Roose-Amsaleg, E. Garnier-Sillam, M. Harry, Extraction
and purication of microbial DNA from soil and sediment
samples, Appl. Soil Ecol. 18 (2001) 4760.
[59] A. Saano, E. Tas, S. Pippola, K. Lindstrom, J.D. Van Elsas,
Extraction and analysis of microbial DNA from soil, in:
J.D. Van Elsas, J.T. Trevors (Eds.), Nucleic Acids in the
Environment: Methods and Applications, Springer-Verlag, ,
Heidelberg, Germany, 1995, pp. 4967.
[60] S. Selenska, W. Klingmller, DNA recovery and direct detec-
tion of Tn5 sequences from soil, Lett. Appl. Microbiol. 13
(1991) 2124.
[61] P. Simonet, A. Capellano, E. Navarro, R. Bardin, A. Moiroud,
An improved method for lysis of Frankia with achromopepti-
dase allows detection of new plasmids, Can. J. Microbiol. 30
(1984) 12921295.
[62] P. Simonet, M.C. Grosjean, A.K. Misra, S. Nazaret, B. Courn-
oyer, P. Normand, Frankia genus-specic characterization by
polymerase chain reaction, Appl. Environ. Microbiol. 57
(1991) 32783286.
[63] R.J. Steffan, J. Goksoyr, A.K. Bej, R.M. Atlas, Recovery of
DNA from soils and sediments, Appl. Environ. Microbiol. 54
(1988) 29082915.
[64] C.C. Tebbe, W. Vahjen, Interference of humic acids and DNA
extracted directly from soil in detection and transformation of
recombinant DNA from bacteria and a yeast, Appl. Environ.
Microbiol. 59 (1993) 26572665.
[65] C.C. Tien, C.C. Chao, W.L. Chao, Methods for DNA extrac-
tion from various soils: a comparison, J. Appl. Microbiol. 86
(1999) 937943.
[66] V. Torsvik, J. Goksoyr, F.L. Daae, High diversity in DNA of
soil bacteria, Appl. Environ. Microbiol. 56 (1990) 782787.
[67] V.L. Torsvik, Isolation of bacterial DNA from soil, Soil Biol.
Biochem. 10 (1980) 1521.
[68] V.L. Torsvik, F.L. Daae, J. Goksoyr, Extraction, purication,
and analysis of DNA from soil bacteria, in: J.D. Van Elsas,
J.T. Trevors (Eds.), Nucleic Acids in the Environment: Meth-
ods and Applications, Springer-Verlag, Heidelberg, Germany,
1995, pp. 2948.
[69] V.L. Torsvik, J. Goksoyr, Determination of bacterial DNA in
soil, Soil Biol. Biochem. 10 (1978) 712.
[70] Y.L. Tsai, B.H. Olson, Rapid method for direct extraction of
DNA from soil and sediments, Appl. Environ. Microbiol. 57
(1991) 10701074.
[71] Y.L. Tsai, M.J. Park, B.H. Olson, Rapid method for direct
extraction of mRNA from seeded soils, Appl. Environ. Micro-
biol. 57 (1991) 765768.
[72] P.E. Turpin, K.A. Maycroft, C.L. Rowlands, E.M. Wellington,
An ion-exchange based extraction method for the detection of
salmonellas in soil, J. Appl. Bacteriol. 74 (1993) 181190.
[73] T. Volossiouk, E.J. Robb, R.N. Nazar, Direct DNA extraction
for PCR-mediated assays of soil organisms, Appl. Environ.
Microbiol. 61 (1995) 39723976.
[74] F. Von Wintzingerode, U.B. Gobel, E. Stackebrandt, Determi-
nation of microbial diversity in environmental samples: pit-
falls of PCR-based rRNA analysis, Fems. Microbiol. Rev. 21
(1997) 213229.
[75] I.G. Wilson, Inhibition and facilitation of nucleic acid ampli-
cation, Appl. Environ. Microbiol. 63 (1997) 37413751.
[76] C.C. Young, R.L. Burghoff, J.G. Keim, V. Minak-Berbero,
J.R. Lute, S.M. Hinton, Polyvinylpyrrolidoneagarose gel
electrophoresis purication of polymerase chain reaction-
ampliable DNA from soils, Appl. Environ. Microbiol. 59
(1993) 19721974.
[77] J. Zhou, M.A. Bruns, J.M. Tiedje, DNA recovery from soils of
diverse composition, Appl. Environ. Microbiol. 62 (1996)
316322.
190 P. Robe et al. / European Journal of Soil Biology 39 (2003) 183190