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1Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina
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We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.
Bey, B. S., Fichot, E. B., Norman, R. S. Extraction of High Molecular Weight DNA from Microbial Mats. J. Vis. Exp. (53), e2887, doi:10.3791/2887 (2011).
Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.
1. Microbial Cell Extraction:
2. DNA Extraction and Purification:
3. DNA Purity, Concentration and Size Determination:
Sequential microbial cell extracts (supernatants) have shown that the turbidity of extracts decreases as the number of extractions increase. This suggests a reduction in the number of cells following each successive cell extraction. Important to note here is that as each additional cell extraction step provides the opportunity for contaminant introduction, the number of cell extractions should be minimized so that the final cell pellet is representative of the overall microbial community. Other sources of contamination were controlled by adopting adequate laboratory sterile techniques. For instance, the solutions were prepared in autoclaved DI water and filter sterilized Containers were sterilized with alcohol, autoclaved, and treated under UV light and ultraviolet crosslinker.
DNA concentration and quality determination:
The protocol yielded approximately 2 μg of HMW DNA (35-50 kb) (Fig. 4) per gram of mat sample with an A260/280 ratio of 1.6, and an A260/230 ratio of 0.7 (Table 1). Although the A260/230 ratio appeared to be low, no inhibition of downstream molecular-based application such as PCR-amplification of the 16S rRNA genes was observed in a separate study7. It is important to note that DNA from hypersaline mats has two main sources of contamination; EPS and salts. It is therefore possible that traces of these contaminants may be influencing the A260/230 ratios despite the tremendous efforts to reduce their effects on down stream applications.
DNA size determination:
Pulse field gel electrophoresis employs a pulsating current flow with intermittent directional switches resulting in a DNA smear as shown in figure 3. Our protocol yielded a HMW DNA of approximately 35-50 kb. While some DNA size may be smaller than 30 kb, it is important to have part of the smear above 35 kb given that fosmid cloning requires ˜40 kb DNA inserts and larger DNA fragments provide greater access to intact biosynthetic pathways. In our studies, the DNA smear above 35 kb was excised and purified for large-insert vector cloning and other molecular applications.
Figure 1. Hypersaline microbial mat sampling site (Big Pond) located on Eleuthera, The Bahamas.
Figure 2. A cross-section of the hypersaline microbial mat used in this study. The microbial mat was obtained from a hypersaline pond located on Eleuthera, The Bahamas.
Figure 3. Schematic representation of procedures involved in microbial cell extraction, cell lysis, extraction, and purification of metagenomic DNA. Click here to view a larger image.
Figure 4. Molecular weight characterization of extracted DNA using pulse field gel electrophoresis. Lane M is a HMW marker, and lanes 1 and 2 are replicates of metagenomic DNA extracted from the Eleuthera hypersaline mat using the above protocol.
|Extraction Method||Concentration ng/g||A260/280||A260/230|
Table 1. Measurement of concentration and quality of DNA extracted from microbial hypersaline mat.
Given that total cell removal from complex and highly diverse microbial mat samples is not practical, the primary concern is how well the extracted cells represent the overall microbial mat community. In a previous study, PCR-DGGE analysis of microbial 16S rRNA genes showed that the five cell removal steps used in this protocol extracts cells that are representative of the overall microbial mat community7. The actual number of cell extraction steps required to provide a cell pellet that is representative of the overall microbial community will likely change dependent upon sample type. For optimal protocol development for different samples, a small-scale pilot study is recommended wherein empirical testing of the microbial richness recovered after each cell extraction step is compared to the richness of the original community.
Fosmid cloning requires HWM DNA of 35-40 kb for clone library construction8. Our protocol yielded DNA with sizes ranging from 35-50 kb (Fig. 4) and was successfully used to generate a fosmid-based metagenomic library (unpublished results). Other protocols used in DNA extraction from EPS-producing marine bacteria yielded ˜23 kb4. In functional metagenomic studies, large DNA fragments provide greater access to a wide range of genes encoding for biosynthetic pathways as well as for functional behaviors 9,10. Thus, HMW DNA is an important requirement for metagenomic studies. Generating large-insert metagenomic libraries from environmental samples will increase the chances of discovering novel biosynthetic pathways that may lead to the discovery of novel genes. This protocol may be used to extract DNA from other refractory environmental samples.
No conflicts of interest declared.
This work was funded by the National Science Foundation Environmental Genomics Program (Grant No. EF-0723707).
|Polyethylene glycol 8000||Promega Corp.||V3011||20% in 1.2 M NaCl|
|Potassium acetate||Fisher Scientific||Fisher Scientific|
|Quant-iT dsDNA Assay kit||Invitrogen||Q33130|
|Sodium Chloride||VWR||BDH8014||Appropriate conc.|
|Sodium Dodecyl Sulfate||Fisher Scientific||03-500-509||10% in water|
|sodium hexametaphosphate||EMD Millipore||SX0583-3||2% in water|
|CHEF Mapper XA System||Bio-Rad||170-3670|
|NanoDrop 1000 spectrophotometer||Thermo Fisher Scientific, Inc.||ND-1000|
|Vortexer||Scientific Industries Inc.|
|Ultraviolet Crosslinker||UVP Inc.|
|Waring blender||Waring Laboratory||LB10S|
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