Articles
Novel High Throughput Method For Studying Microbial Communities And Genes In Soil
Tue, 2024-06-24 06:14
Soil represents one of the most diverse habitats for microorganisms.
by Ravi J. Venugopal and Mark N. Brolaski
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.
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).
by Ravi J. Venugopal and Mark N. Brolaski
The UltraClean-htp 96 Well Soil DNA Kit |
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. | 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. | 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. |
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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