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

doi:10.1007/s11783-014-0679-4

Front. Environ. Sci. Eng.

2015, Vol. 9

Issue (3) : 444-452 https://doi.org/10.1007/s11783-014-0679-4

RESEARCH ARTICLE

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

Yun FANG^1,^2,^3,⁴,Meiying XU^1,^4,^*(

),Xingjuan CHEN^1,⁴,Guoping SUN^1,⁴,Jun GUO^1,⁴,Weimin WU⁵,Xueduan LIU^2,^3,^*(

)

1. Guangdong Institute of Microbiology, Guangzhou 510070, China
2. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China
3. Key Laboratory of Biometallurgy of Ministry of Education, Changsha 410083, China
4. State Key Laboratory of Applied Microbiology (Ministry-Guangdong Province Jointly Breeding Base), Guangzhou 510070, China
5. Department of Civil & Environmental Engineering, Center for Sustainable Development & Global Competitiveness, Stanford University, Stanford, CA94305, USA

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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

Corresponding Author(s): Meiying XU,Xueduan LIU

Online First Date: 14 March 2014 Issue Date: 30 April 2015

Cite this article:

Yun FANG,Meiying XU,Xingjuan CHEN, et al. Modified pretreatment method for total microbial DNA extraction from contaminated river sediment[J]. Front. Environ. Sci. Eng., 2015, 9(3): 444-452.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-014-0679-4
https://academic.hep.com.cn/fese/EN/Y2015/V9/I3/444

Tab.1 Comparison of DNA yield and purity of the crude DNA with different pretreatment parameters

Fig.1 Agarose gel electrophoresis of the total crude DNA extracted from the sediment samples with different mass. M: λDNA/HindIII marker; lane 1 and 2: 0.25 g; lane 3 and 4: 0.12 g; lane 5 and 6: 0.06 g; lane 7 to 10: 0.03 g

Fig.2 Agarose gel electrophoresis profiles of the total crude DNA extracted with different pretreatment temperatures. M: λDNA/HindIII marker; lanes 1 to 3: 25°C; lane 4 to 6: 37°C; lane 7 to 9: 50°C; lane 10 to 12: 65°C

Fig.3 Agarose gel electrophoresis profiles of the restriction enzyme digestion products of crude DNA samples extracted with different pretreatment temperatures. M: λDNA/HindIII marker

Fig.4 Agarose gel electrophoresis profiles of the PCR products of V3 region of 16S rRNA gene from the crude DNA samples extracted with different methods. (a) DNA samples extracted with different pretreatment temperatures, and (b) DNA samples extracted with our modified method (lane 1 and 2) versus with the traditional standard extraction method (lane 3 and 4). M: DS 5000 marker. CK: control treatment (without a DNA template)

Fig.5 DGGE gel profiles of the DNA samples with different extraction methods. Lane 1: with a PowerSoil^TM DNA Isolation Kit; Lanes 2 and 3: with our proposed method with pretreatment

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