Mo Bio Laboratories, Inc.
2746 Loker Ave. West
Carlsbad, CA, 92010
Website: http://www.mobio.com
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Novel High Throughput Method For Studying Microbial Communities And Genes In Soil
Soil represents one of the most diverse habitats for
microorganisms.
by Ravi J. Venugopal and Mark N.
Brolaski
The UltraClean-htp 96 Well Soil DNA Kit
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Introduction
High quality genomic DNA extracted directly from
soil, water and sediment samples is essential to examine molecular phylogenetic
diversity and to understand and exploit the functional diversity of
microorganisms. To date, the recovery of bacterial DNA from complex environments
has been achieved either by direct extraction of DNA from soil (in situ lyses of
bacteria) or from pre-isolated (but uncultivated) bacteria. Comparison of these
two techniques has shown that although there are some variations in the amount
and quality of DNA recovered, no major phylogenetic bias is observed.(1) The
major concern with these conventional methods is the time they require, from 2
to 3 days, to extract high quality DNA. With respect to this problem, the
quality of the DNA may not be always be sufficient for use in downstream
applications. Furthermore, samples are processed one at a time, making the
actual process of DNA extraction slow and cumbersome. As a consequence,
researchers have been limited in their ability to exploit the wealth of
information present in the microbial communities of natural
environments.
The problem of low quality DNA directly extracted from
soil samples is a result of the DNA fraction being negatively compromised by
co-purified contaminants. For example, both clay and organic fractions of soil
affect DNA isolation and purification differently.(2) Clay has a tendency to
bind DNA adsorptively, where as humic polymers found in the organic fraction
tend to co-purify with DNA during the extraction process. Humic acids pose a
considerable problem and will interfere in enzymatic manipulations of DNA and
inhibit the polymerase enzyme used in PCR. Some of these compounds also appear
to co-migrate with DNA during CsCl-ethidium bromide isopycnic
ultracentrifugation, resulting in light brown coloration of the recovered
DNA.(3) These observations imply an intimate association between the
contaminants and the DNA.
A diverse habitat
Both culture-based and culture-independent approaches
support the hypothesis that soil represents one of the most diverse habitats for
microorganisms.(4) Molecular based studies have confirmed soil as an environment
particularly rich in diversity by comparing 16S ribosomal RNA sequences from
several divergent bacterial divisions such as a,b,g, and d Proteobacteria.
Despite these findings, the extent of microbial diversity in nature is still
largely unknown. This insight provides the scientific foundation for a renewed
interest in examining soil microorganisms for novel pharmaceuticals and has
inspired the development of approaches to access the metabolic potential of soil
microorganisms without culturing them.(5)
Microbial genomic DNA
extracted directly from the soil matrix can be inserted into vectors propagated
in bacterial strains such as E. coli, that are easy to grow and amenable to
genetic manipulations. Using this method, it is possible to access the entire
genome of an ecosystem by creating gene libraries. Environmental DNA libraries
that recover functional genes from uncultivated bacteria provide a promising
drug discovery tool. The majority of more than 5000 known anti-infective
compounds are natural product derivatives, with over 100 in clinical use.(6)
Furthermore, natural products are the main source of cancer chemotherapeutics,
immuno-modulating compounds, and other pharmaceutical molecules.(7) The
potential to perform high throughput isolation of DNA from soils in a relatively
short time without co-purified contaminants would greatly facilitate access to
the genomes of microorganisms in natural environments and speed up the drug
discovery process.
A high throughput method for soil DNA extraction
To address these
significant problems, Mo Bio Laboratories, Incorporated, (Solana Beach,
California) has developed a high throughput method for soil DNA extraction. The
UltraClean-htpTM 96 Well Soil DNA Kit has the ability to isolate high quality DNA
from 96 samples in less than 1.5 hours. The reagents in the kit allow for
co-purified contaminants to be removed during processing. This ensures
extraction of DNA pure enough for PCR amplification, restriction enzyme
digestion and other downstream molecular techniques.
A multi-pronged
approach is employed. A proprietary reagent, IRS-Inhibitor Removal Solution,
addresses the issue of contaminants co-purifying with the DNA. When mixed with
the soil samples, this solution effectively precipitates all humic acids and
allows for the DNA to be used in enzymatic reactions like PCR. The other major
development utilized in this kit is bead-beating technology formatted in 96 well
plates. Soil samples are added to wells containing specialized beads and
solutions. Cells of all types are lysed by shaking at high velocities. The
shaker recommended for this procedure is the Retsch (Newtown, Pennsylvania)
Model MM301. It allows for processing two 96 well plates at one time. As the
cells are lysed, nucleic acids are released into solution. Nucleic acids are
then captured using silica spin filters in a 96 well plate format. After binding
and washing the filters, the genomic DNA is eluted into certified DNA-free Tris
solution. By using this methodology, high quality DNA can be isolated from soil
samples in a very short period of time.
Figure 1: Genomic DNA extracted from 12 different soil samples. DNA was
separated by electrophoresis on a 1.2%, 1 TAE agarose gel and visualized by
ethidium bromide staining. Lane 1 is a 1 kb DNA ladder and lanes 2 through13
represent 12 different soil samples.
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Figure 2: PCR products obtained for different groups of microorganisms from
two different types of soil samples. Products are separated by electrophoresis
on a 1.2%, 13 TAE agarose gel and visualized by ethidium bromide staining. (1
& 14) 1 kb DNA ladder, (2) Negative control, (3) Positive control, (4 &
9) Eubacteria, (5 & 10) Plant, (6 &11) Fungi, (7 & 12) Streptomyces,
(8 & 13) Bacilli.
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Figure 3: PCR products obtained for three different genes from two different
types of soil samples. Products were separated by electrophoresis on a 1.2%, 13
TAE agarose gel and visualized by ethidium bromide staining. (1) A 1 kb ladder
DNA molecular weight marker, (2) Negative control, (3) Positive control, (4
& 7) Chloroplast gene rbcL, (5 & 8) Nitrogenase gene nifH, (6 & 9)
Heat shock protein gene hsp70.
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Effectiveness and efficiency
To establish the effectiveness and efficiency
of the UltraClean-htp 96 Well Soil DNA Kit, two parameters were reviewed. First,
the consistency in DNA yield across the 96 well plate was evaluated. Using
garden soil as a standard, the 96 well prep yielded an average of 6.46 mg DNA
per 250 mg of wet soil sample. Minimal yield variation was observed across all
the wells on the plate. Among 48 different soil and sediment samples that have
been tested, the DNA yield ranged from 1.6 mg per 250 mg (wet weight) to 9 mg/
per 250 mg (wet weight) (data not provided). The DNA quantity of the soil
samples is largely a function of soil type, and thus the yield is expected to
vary from sample to sample. Figure 1 shows DNA extracted from a set of twelve
samples using the UltraClean-htp 96 Well Soil DNA Kit.
The second
parameter reviewed was the quality of DNA obtained using the UltraClean-htp 96
Well Soil DNA Kit. This parameter was initially evaluated by analyzing the
A260/280 UV absorbance ratio of the DNA. DNA samples extracted with the kit
demonstrated acceptable ratios of absorbance at 260 nm and 280 nm being in the
range of 1.8 to 2.0 indicating that this is a pure DNA sample free from protein,
polyphenols or lipid contaminants.(4)
In addition to testing purity,
DNA samples extracted with this kit were evaluated in a functional test
utilizing the PCR reaction. This test was utilized to demonstrate the absence of
co-purified humic acid contaminants. A group of primer sets representative for
the genomes of Eubacteria, plant, fungi, Streptomyces, and Bacilli were used in
the reactions. PCR products were successfully obtained with DNA isolated from
two different soil types as shown in Figure 2. In addition, by including a set
of commercially important genes (bacterial hsp70, nifH and plant chloroplast
gene, rbcL, we demonstrate the feasibility of high throughput in situ analyses
of genes directly in soil samples, as shown in Figure 3.
Summary
In summary, the development of the UltraClean-htp 96 Well Soil DNA
Kit should offer researchers an efficient high throughput method to study the
microbial communities and genes in soil. By obtaining high quality DNA free of
contaminants, molecular methods such as PCR, restriction digests, Southern
blotting, cloning and other downstream applications can be performed with the
highest level of confidence. This kit provides basic and pharmaceutical
researchers the opportunity to utilize direct molecular techniques when
analyzing the most important groups of microorganisms present in soils.
Therefore, the UltraClean-htp 96 Well Soil DNA Kit is a suitable method for
efficient and accurate analyses of microbial community structure in natural
environments.
About the authors
Ravi J. Venugopal is Director of Research and
Development, and Mark N. Brolaski is President, both with Mo Bio Laboratories,
Incorporated.
More information about extraction of DNA from soil is available
from: Mo Bio Laboratories, Inc., Carlsbad, CA. 800-606-6246; mobio.com
References
1. Courtois, S., Frostegard, A., Goransson, P., Depret, G.,
Jeannin, R. and Simonet, P. Environ. Microbiol. 3:431-439 (2001).
2. Ogram,
A.V., Sayler, G.S., Gustin, D. and Lewis, R.J. Environ. Sci. Tech. 22:982-984
(1987).
3. Ogram, A., Sayler, G.S., and Barkay, T. J. Microbiol. Methods.
7:57-66 (1988).
4. Hugenholtz, P., Goebel, B.M. and Pace, N.R. J. Bacteriol.
180:4765-4774 (1998).
5. Rondon, M.R., Goodman, R.M., and Handelsman, J.
Trends Biotechnol. 17:403-409 (1999).
6. Harvey, A. 2000. Drug Discovery
Today. 5:294-300 (2000).
7. Cragg, G.M. and Newman, D.J. Expert Opin.
Investig. Drugs. 9:2783-2797 (2000).
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