Transcript Advanced Environmental Biotechnology II

Advanced Environmental
Biotechnology II
Nucleic acids from the environment
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.
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.
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 then DNA extraction and
purification.
The major concern with the so-called indirect or
ex situ DNA extraction approach is differences in
how 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 co-extracted 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.
DNA extraction seems to work reliably for
different samples, but efficient RNA extraction
often has problems.
Recovery of cells from environmental samples.
The indirect DNA extraction approach might
preferably be used when problematic
environmental matrices are to be analyzed, 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 protocols aiming at the representative
dislodgement and extraction of surface-attached
cells have been published. All these methods
have in common that they use repeated
homogenization and differential centrifugation.
The protocols differ considerably with respect to
the solutions used to break up soil colloids and
dislodge surface-attached cells that adhere to
surfaces by various bonding mechanisms such as
polymers, electrostatic forces and water
bridging, and that act to differing degrees.
Homogenization is usually achieved by shaking
suspensions with gravel or blending in
Stomacher or Waring blenders. In particular, for
soils with a high clay content a further
purification can be achieved by sucrose/Percoll
density gradient centrifugation or flotation of
the bacterial fraction on a Nycodenz cushion
Although a complete dislodgement of cells
seems to be impossible, 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.
Cell lysis and DNA extraction protocols
The efficient disruption of the bacterial and
fungal cell walls is crucial for the recovery of
representative 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 disintegration of cell
walls.
The efficiency of cell lysis protocols might differ
considerably depending on the kind of
environmental matrix, since compounds within
the matrix might have adverse effects, e.g.
reduced enzyme activity due to non-optimal pH
or ionic conditions, or simply due to a high
adsorption capacity. A number of studies
compared the efficiency of lysis protocols with
respect to DNA yield and fragmentation.
In most studies, bead beating was said to give
the highest amounts of DNA, but the DNA
produced was broken to some degree.
Grinding in the presence of liquid nitrogen might
be helpful.
Bead beating is an important step before
treatment with other cell lysis methods.
The efficiency of the cell lysis is usually
estimated by microscopic examination. A
correlation of lysis efficiency and clay content
was demonstrated.
Common lysis approaches such as bead beating
and also three different extraction and
purification protocols have been compared.
Comparison of four common lysis procedures on the yield and
fragmentation of the DNA’
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).
The use of commercial kits takes less time and
avoids extraction with phenol and chloroform.
The DNA extracted with the commercial kits less
frequently contained PCR-inhibiting substances.
The use of small amounts of soil (0.25–0.5 g) for
DNA extraction may be a serious limitation for
the recovery of a sufficient quantity of DNA to
be representative of that environment.
The yield of DNA recovered per gram of soil
depends on the lysis method and on the
extraction protocol used.
Tthe soil type strongly affected the quality
(degree of shearing, PCR-inhibiting 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 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 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.
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.
The majority of bacteria are thought to take up
and incorporate [3H] thymidine into DNA, but
this has not been fully investigated for its
analogue BrdU.
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.
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.
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.