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Extraction of high molecular weight DNA from microbial mats.
Biotechniques. 2010 Sep;49(3):631-640
Authors: Bey B, Fichot E, Dayama G, Decho A, Norman RS
Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields >2 µg HMW DNA (>48 kb) per gram of mat sample, with A260:280 ratios >1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness. Address correspondence to R. Sean Norman, University of South Carolina, Department of Environmental Health Sciences, 921 Assembly St., PHRC 401, Columbia, SC 29208, USA. e-mail:
PMID: 20854264 [PubMed - as supplied by publisher]
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Extraction of high molecular weight DNA from microbial mats.
Biotechniques. 2010 Sep;49(3):631-640
Authors: Bey B, Fichot E, Dayama G, Decho A, Norman RS
Due to the presence of inhibitors such as extracellular polymeric substances (EPSs) and salts, most microbial mat studies have relied on harsh methods of direct DNA extraction that result in DNA fragments too small for large-insert vector cloning. High molecular weight (HMW) DNA is crucial in functional metagenomic studies, because large fragments present greater access to genes of interest. Here we report improved methodologies for extracting HMW DNA from EPS-rich hypersaline microbial mats. The protocol uses a combination of microbial cell separation with mechanical and chemical methods for DNA extraction and purification followed by precipitation with polyethylene glycol (PEG). The protocol yields >2 µg HMW DNA (>48 kb) per gram of mat sample, with A260:280 ratios >1.7. In addition, 16S rRNA gene analysis using denaturing gradient gel electrophoresis and pyrosequencing showed that this protocol extracts representative DNA from microbial mat communities and results in higher overall calculated diversity indices compared with three other standard methods of DNA extraction. Our results show the importance of validating the DNA extraction methods used in metagenomic studies to ensure optimal recovery of microbial richness. Address correspondence to R. Sean Norman, University of South Carolina, Department of Environmental Health Sciences, 921 Assembly St., PHRC 401, Columbia, SC 29208, USA. e-mail:
PMID: 20854264 [PubMed - as supplied by publisher]
More...