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^TM 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^TM 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.

Keywords

river sediment / DNA extraction / contaminant / pretreatment / microbial community

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Acknowledgements

The authors thank Yinghua Cen and Xian Fu for their suggestions during manuscript preparation. The authors thank Stephanie Bachas-Daunert for her helps in English language modifications. This research was supported by the National Basic Research Program of China (Grant No. 2012CB22307), the National Natural Science Foundation of China (Grant No. 31170470), Guangdong Provincial Natural Science Foundation of Research Team Program (9351007002000001), the International Cooperation Projects of Guangdong Province (2011B050400005) and Guangdong Provincial Programs for Science and Technology Development (2012A061100009). No conflict of interest exits in this manuscript.Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11783-014-0679-4 and is accessible for authorized users.

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