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Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment Abstract Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol·L−1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol·L−1 Tris (pH 8.0), 1.5 mol·L−1 NaCl, 0.1 mol·L−1 NaH2PO4, and Na2HPO4 at 65°C with 180 r·min−1 for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil™ DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil™ DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png "Frontiers of Environmental Science & Engineering" Springer Journals

Modified pretreatment method for total microbial DNA extraction from contaminated river sediment

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References (31)

Publisher
Springer Journals
Copyright
2014 Higher Education Press and Springer-Verlag Berlin Heidelberg
ISSN
2095-2201
eISSN
2095-221X
DOI
10.1007/s11783-014-0679-4
Publisher site
See Article on Publisher Site

Abstract

Abstract Extraction of high-quality microbial DNA from contaminated environmental samples is an essential step in microbial ecological study. Based on previously published methods for soil and sediment samples, a modified pretreatment method was developed for extracting microbial DNA from heavily contaminated river sediment samples via selection of optimal pretreatment parameters (i.e., reagent solution, reaction duration, and temperature). The pretreatment procedure involves washing the river sediment sample for three times with a solution containing 0.1 mol·L−1 ethylene diamine tetraacetic acid (EDTA), 0.1 mol·L−1 Tris (pH 8.0), 1.5 mol·L−1 NaCl, 0.1 mol·L−1 NaH2PO4, and Na2HPO4 at 65°C with 180 r·min−1 for 15 min to remove humic materials and heavy metals prior to the employment of standard DNA extraction procedures. We compared the results of standard procedure DNA extraction following pretreatment, without pretreatment, and with using a commercial PowerSoil™ DNA Isolation Kit. The results indicated that the pretreatment significantly improved the DNA quality based on DNA yield, DNA fragment length, and determination of prokaryotic diversity. Prokaryotic diversity exhibited in the DNA with the pretreatment was also considerably higher than that extracted with the PowerSoil™ DNA Isolation Kit only. The pretreatment method worked well even with a small amount of sediment sample (0.25 g or even lower). The method provides a novel, simple, cost-effective tool for DNA extraction for microbial community analysis in environmental monitoring and remediation processes.

Journal

"Frontiers of Environmental Science & Engineering" – Springer Journals

Published: Jun 1, 2015

Keywords: Environment, general

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