Advanced Environmental Biotechnology 2
1 Nucleic acid extraction from environmental samples
Annett Milling, Newton C M.Gomes, Miruna Oros-Sichler,
Monika Götz and Kornelia Smalla
Molecular Microbial Ecology BIOS Advanced Methods. (2005) Osborn, A. Mark.; Smith,
Cindy J. Eds. Taylor & Francis Routledge
1.1 Introduction
Recently molecular tools such as nucleic acid hybridization, the polymerase chain
reaction (PCR) and DNA cloning and sequencing became increasingly available. More
attention was focused on the analysis of DNA extracted from environmental bacteria
without prior cultivation. The analysis of nucleic acids extracted directly from
environmental samples allows the researcher to investigate microbial communities by
avoiding the limitations of cultivation techniques. Only a small proportion of bacteria can
form colonies when traditional plating techniques are used. Also, under environmental
stress bacteria can enter a state termed “viable but non-culturable” (vbnc), so they can’t
be grown using traditional cultivation techniques. So researchers thought it would be
good to study nucleic acids directly from environmental samples. This would be
representative of the microbial genomes in the samples. The analysis of DNA can give
information on the structural diversity of environmental samples, or on the presence or
absence of certain functional genes (e.g. genes giving xenobiotic biodegradative
capabilities, antibiotic resistance or plasmid-borne sequences), or to monitor the fate of
bacteria (including genetically modified organisms) released into an environment.
However, in general the analysis of DNA does not allow conclusions to be drawn on the
metabolic activity of members of the bacterial or fungal community or on gene
expression. This information might be obtained from analysis of RNA (rRNA or mRNA).
There are many different ways to get nucleic acids from environmental samples. There
are two main approaches, each with their own advantages and limitations. The first is
based on direct or in situ lysis of microbial cells in the presence of the environmental
matrix (e.g. soil or sediments), followed by separation of the nucleic acids from matrix
components and cell debris. This method is by far the most used. The advantage of the
direct nucleic acid extraction approach is that it takes less time and that a much higher
DNA yield is achieved. However, directly extracted DNA often contains considerable
amounts of co-extracted substances such as humic acids that interfere with later
molecular analysis. Also, a large proportion of directly extracted DNA might come from
non-bacterial sources or from free DNA.
In the second approach, the microbial fraction is separated from the environmental matrix
before cell lysis and subsequent DNA extraction and purification. The major concern
with the so-called indirect or ex situ DNA extraction approach is differences in how
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easily surface-bound cells can be separated. Dissociation of cells from surfaces is usually
done by repeated blending/homogenization steps and differential centrifugation. So the
indirect method is more time-consuming and prone to contamination. An advantage of
the indirect approach is that the nucleic acids recovered are less contaminated with coextracted humic acids and DNA of non-bacterial origin. Researchers are developing
faster and more efficient nucleic acid extraction procedures. The first protocols for both
approaches all required CsCl/ethidium bromide density gradient ultracentrifugation to
purify the DNA and were rather tedious and time-consuming. Using DNA purification
kits based on different kinds of resins considerably improved the purification efficiency
and reduced the time needed to obtain DNA suitable for molecular analysis. However,
none of the protocols was suitable for all soil types, in particular for soils and sediments
coming from contaminated sites. Only recently have commercial kits for DNA extraction
from soils become available. These simplify and miniaturize the method. Commercial
soil DNA extraction kits can be used to extract DNA directly from environmental
samples and/or from the microbial pellet obtained after efficiently removing the microbes
from the sample and then centrifuging. Commercial kits for nucleic acid extraction are
easy to use, but a number of things remain that influence the quantity and quality of
nucleic acid extracts. Whilst DNA extraction seems to work reliably for different
matrices, efficient RNA extraction often has problems. So we will also look at protocols
that let us assess the metabolically active microbial fraction within an environmental
sample. We will study the use of 16S/18S rDNA-based molecular fingerprints for
comparing different protocols and for showing how different methods affect the
microbial community recovered.
1.2 Recovery of cells from environmental matrices
The indirect DNA extraction approach might best be used when there are problems with
the environmental matrices, or when cloning large DNA fragments [e.g. to generate
bacterial artificial chromosome (BAC) libraries] from soil or sediment DNA where a high
proportion of DNA of bacterial origin is crucial. Different ways for the extraction of
surface-attached cells have been published. All these methods have in common that they
use repeated homogenization and differential centrifugation. However, the protocols are
very different with respect to the solutions used to break up soil colloids and dislodge
surface-attached cells that stick to surfaces by various bonding mechanisms such as
polymers (e.g. exopolysaccharides or fimbriae), electrostatic forces and water bridging.
Homogenization is usually achieved by shaking suspensions with gravel or blending in
Stomacher or Waring blenders. Although it may be impossible to unstick all the cells, it
is important that cells that are bound to the surface with different degrees of strength are
released with similar efficiency. This can easily be evaluated by using DNA
fingerprinting, e.g. denaturing gradient gel electrophoresis to analyze 16S or 18S rDNA
fragment profiles amplified from the DNA extracted from the microbial pellet in
comparison to profiles generated from directly extracted DNA.
1.3 Cell lysis and DNA extraction protocols
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The bacterial and fungal cell walls must be broken so we can get the DNA which reflects
the genomes of microbes present in an environmental sample and their relative
abundance. Cell lysis can be achieved by mechanical cell disruption and/or by enzymatic
or chemical breaking of cell walls. Most protocols include a combination of these steps.
The efficiency might differ considerably depending on the kind of environmental matrix,
since compounds within the matrix might have bad effects, e.g. reduced enzyme activity
due to non-optimal pH or ionic conditions, or simply due to a high adsorption capacity. In
most studies, bead beating yielded the highest amounts of DNA, although the DNA
produced showed some degree of shearing. Some researchers like grinding in the sample
in liquid nitrogen.
Figure 1.1
Comparison of four common lysis procedures with respect to the yield and fragmentation
of the DNA (0.8% agarose gel). Lane M: 1 kb ladder; lane 1: bead beating; lane 2: Retsch
mill; lane 3: vortex; lane 4: freeze-boiling cycles (20 min at 63°C followed by 20 min at
−20°C).
While the quantity and degree of nucleic acid fragmentation was assessed by agarose gel
electrophoresis, the influence of the different protocols on the bacterial and fungal
diversity was evaluated by denaturing gradient gel electrophoresis (DGGE) analysis of
16S/18S rDNA fragments amplified from the different kinds of DNA. In contrast to
traditional DNA extraction protocol, the use of commercial kits is less time-consuming
and avoids extraction with phenol and chloroform. In addition, the DNA extracted with
the commercial kits less frequently contained PCR-inhibiting substances. A disadvantage
may be that the use of rather small amounts of soil (0.25–0.5 g) for DNA extraction, from
some environmental matrices, may represent a serious limitation for the recovery of a
sufficient quantity of DNA to be representative of that environment. The amount of DNA
recovered per gram of soil depends on the lysis method and on the extraction protocol
used. In addition, the soil type strongly affected the quality (degree of shearing, PCRinhibiting substances) and the quantity of the DNA. Although the quantification of DNA
based on agarose gels stained with DNA-staining dyes is more complicated than
fluorimetric measurements, this approach also provides insights into the degree of DNA
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shearing. High molecular weight DNA is an important criterion when evaluating and
comparing different protocols because sheared DNA can cause PCR artifacts and is not
suitable for direct cloning of large DNA fragments. DNA yields reported in the literature
on soil range from 5 to 250 µg g−1 of soil.
The complete removal of co-extracted humic acids is needed. Humic acids were shown to
interfere with DNA hybridization, restriction enzyme digestions and PCR amplification.
The amount of humic substances strongly depends on the soil type. DNA extracted using
commercial kits is often suitable for use in PCR amplifications without additional
purification steps, except when applied to sandy soils. A range of methods has been used
to remove co-extracted substances, e.g. silica-based or ion exchange resins.
1.4 Immunochemical isolation of DNA from metabolically active cells
Uptake of [3H]thymidine has routinely been used for measuring the in situ growth of
bacteria in different environments. Bromodeoxyuridine (BrdU) is structurally similar to
thymidine, and since it can be incorporated into newly synthesized DNA it is widely used
in medical research. Recently, researchers have shown the potential use of BrdU to detect
metabolically active bacteria in microbial communities from lake water, bacterioplankton
or in soil. The procedure consists of four steps:
(i) incubation of environmental bacteria (soil or microbial fraction) with BrdU;
(ii) extraction of DNA directly from the environmental sample or the microbial fraction;
(iii) immunocapture of DNA containing incorporated BrdU by using magnetic beads
covered with anti-BrdU antibody;
(iv) immunoprecipitation.
The BrdU method was used to study soil bacterial communities responding to
environmental stimuli such as the addition of glucose. Soils with or without glucose
amendment were mixed with BrdU and incubated at room temperature.
A potential limitation of the BrdU method might be the proportion of bacteria that are
capable of incorporating BrdU. Whilst the majority of bacteria are thought to take up and
incorporate [3H] thymidine into DNA, this has not been fully investigated for its analogue
BrdU.
1.5 RNA extraction from environmental matrices
Methods for RNA extraction have been less frequently used and are less well known.
Due to the short half-life of bacterial messenger RNA as well as the high abundance and
persistence of RNases, an unbiased recovery of total RNA is difficult. Considerable effort
is required to ensure the absence of RNases.
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Different protocols aimed at the simultaneous extraction of RNA and DNA have recently
been published. As with DNA extractions, important criteria for the quality and
suitability of RNA extraction protocols are the yield, the integrity, and the purity of the
RNA. Co-extracted humic substances and DNA have been reported to affect RNA
hybridization results with oligonucleotide probes. Probe hybridization decreased as the
concentration of DNA or humic substances increased. Humic substances and/or high
concentrations of co-extracted DNA were shown to result in membrane saturation and
consequently reduced amounts of bound RNA. Even the presence of low concentrations
of DNA seemed to cause a reduced accessibility of the rRNA target. Another major
concern is that some DNase preparations might contain residual RNase activity and cause
partial degradation of rRNA molecules. rRNA is intact when distinct bands of the small
and large subunit RNA can be seen after electrophoresis (Figure 1.2, lane A1).
Different regions of the ribosomal RNA are differentially susceptible to the attack of
RNases. Such partial RNA degradation is particularly critical when the RNA is used for
quantitative hybridization. Methods for efficiently removing humic substances and
residual DNA without partial degradation of the RNA are required for the reliable use of
RNA for microbial community analysis. While the size and amount of rRNA can be
assessed by polyacrylamide or agarose gels, the presence of mRNA is usually confirmed
by PCR amplification or by membrane hybridization with probes.
Figure 1.2
Agarose gel electrophoresis of nucleic acids recovered from bulk soil. Comparison of a
classical extraction method and commercial kits for extraction and purification of soil
DNA. M: 1 kb DNA marker; A1: crude DNA; A2: two-step purified DNA; A3: DNA
purified with the Ultra Clean™ 15; A4: DNA purified with the Geneclean Spin® Kit; B:
DNA extracted with the Ultra Clean™ Soil DNA Kit; C: DNA extracted with the Fast
DNA Spin® Kit.
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