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Authors: Patrick J. Cummings, Kristina M. Obom​

Citation: Patrick J. Cummings, Kristina M. Obom. 2008. Metagenomic soil DNA analysis.​

Publication Date : August 2008​

 

Introduction ​

FIG. 1. Agarose gel analysis of metagenomic DNA. Metagenomic DNA (multiple genomic DNAs) was isolated from different soil samples and then separated by 0.7% agarose gel electrophoresis and stained with ethidium bromide. Ethidium bromide dye intercalates within DNA strands and allows for visualization upon exposure to ultraviolet light. Note the large molecular weight genomic DNA demonstrating different quantities (amount of fluorescent high molecular DNA, compare lane 6 to lane 7) and quality of genomic DNA (compare lane 1 to lane 2). Lanes 1 through 7, 5 ul of genomic DNA isolated from different soil samples; lane 8, molecular weight marker, lambda phage DNA digested with HindIII restriction enzyme. ​

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FIG. 2. Polymerase chain reaction (PCR) of metagenomic DNA.  Results of metagenomic DNA amplified by PCR using Bacillus subtilis rRNA primers (6) and analyzed by agarose gel electrophoresis. Note different levels of rRNA amplicons (595 base pairs) from three different soil samples (lanes 2, 4, and 5). Amplicons are portions of gene sequences copied by amplification technologies such as PCR. Lane 3, Bacillus subtilispositive control. Lane 1, 2 Kb molecular weight marker; lane 6, molecular weight marker lambda phage DNA digested with HindIII restriction enzyme.  ​

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Methods ​

Using the PowerMax Soil DNA Isolation Kit (MO BIO Labs, Inc.), 10 grams of soil were processed for metagenomic DNA purification.  Bacterial lysis occurred by mechanical (vortexing) and chemical methods, followed by capture of genomic DNA on a silica membrane. Eluted DNA was precipitated and then washed with 70% ethanol. DNA was dissolved in 150 ul of sterile water and then analyzed by agarose gel electrophoresis (Fig. 1).   ​

PCR of genomic DNA was performed using primers specific to the 16S rRNA B. subtilis group (6). Using 1 ug of genomic DNA, 25 pmol of primers, and puReTaq Ready-To-Go PCR beads (Amersham Biosciences), samples were treated to the following cycling profile: 95oC for 0.5 min., 65oC for 2 min., and 72oC for 2 min. for 30 cycles (Fig. 2). ​

Metagenomic DNA analysis involves the isolation and sequencing of microbial genetic information from complex systems such as the natural environment or host tissues. Specific fields of study that utilize metagenomic investigations include environmental gene surveys, noncultivatable microbe characterization, bacterial community metabolism, microbial diversity and evolution, microbial ecology, and bioremediation (1, 2, 4). ​

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Discussion  ​

The use of biological agents as weapons could result in mass casualties. The "anthrax letter" attacks of 2001 demonstrated the potential for this method of attack by terrorist organizations (3). Biological agents can be used to contaminate various environmental matrices such as air, water, soil, and fomites. Soil is a known vehicle for transmission of the category A agents, Bacillus anthracis, Francisella tularensis, and hantavirus. Moreover, B. anthracis, Yersinia pestis, F. tularensis, hantavirus, and Marburg virus occur in feces and sewage following naturally occurring outbreaks (5). Determining the survival and persistence of select agents in the environment has an essential role in their detection, biocontainment, and subsequent bioremediation efforts.   ​

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References  ​

1. Allen, E. E., and J. F. Banfield.   2005. Community genomics in microbial ecology and evolution.   Nat. Rev. Microbiol. 3:489–498. ​

2. Breitbart, M., P. Salamon, B. Andresen, J. M. Mahaffy, A. M. Segall, D. Mead, F. Azam, and F. Rohwer. 2002. Genomic analysis of uncultured marine viral communities. Proc. Natl. Acad. Sci. USA 99 :14250–14255. ​

3. Dewan et al. 2002. Inhalational anthrax outbreak among postal workers, Washington, D.C. 2001. Emerg. Infect. Dis. 8c:1066 – 1072.   ​

4. Eisen, J. A. 2007. Environmental shotgun sequencing: its potential and challenges for studying the hidden world of microbes. PLoS Biol. 5(3):e82.  http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0050082.   ​

5. S inclair et al. 2008. Persistence of category A select agents in the environment. Appl. Environ. Microbiol. 74 :555 – 563. ​

6. Wattiau et al. 2001. A PCR test to identify Bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment. Appl. Microbiol. Biotechnol. 56:816 – 819. ​

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