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Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

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Abstract

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0). Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed: one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.

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References

  1. HAMADY M, LOZUPONE C, KNIGHT R. Fast uniFrac: Facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data [J]. Isme Journal, 2010, 4(1): 17–27.

    Article  Google Scholar 

  2. MARGULIES M, EGHOLM M, ALTMAN W E, ATTIYA S, BADER J S, BEMBEN L A, BERKA J, BRAVERMAN M S, CHEN Y J, CHEN Z T, DEWELL S B, DU L, FIERRO J M, GOMES X V, GODWIN B C, HE W, HELGESEN S, HO C H, IRZYK G P, JANDO S C, ALENQUER M L I, JARVIE T P, JIRAGE K B, KIM J B, KNIGHT J R, LANZA J R, LEAMON J H, LEFKOWITZ S M, LEI M, LI J, LOHMAN K L, LU H, MAKHIJANI V B, MCDADE K E, MCKENNA M P, MYERS E W, NICKERSON E, NOBILE J R, PLANT R, PUC B P, RONAN M T, ROTH G T, SARKIS G J, SIMONS J F, SIMPSON J W, SRINIVASAN M, TARTARO K R, TOMASZ A, VOGT K A, VOLKMER G A, WANG S H, WANG Y, WEINER M P, YU P G, BEGLEY R F, ROTHBERG J M. Genome sequencing in microfabricated high-density picolitre reactors [J]. Nature, 2005, 437(7057): 376–380.

    Google Scholar 

  3. TRINGE S G, RUBIN E M. Metagenomics: DNA sequencing of environmental samples [J]. Nature Reviews Genetics, 2005, 6(11): 805–814.

    Article  Google Scholar 

  4. WU L Y, THOMPSON D K, LI G S, HURT R A, TIEDJE J M, ZHOU J Z. Development and evaluation of functional gene arrays for detection of selected genes in the environment [J]. Applied and Environmental Microbiology, 2001, 67(12): 5780–5790.

    Article  Google Scholar 

  5. THAKURIA D, SCHMIDT O, MAC SIURTAIN M, EGAN D, DOOHAN F M. Importance of DNA quality in comparative soil microbial community structure analyses [J]. Soil Biology & Biochemistry, 2008, 40(6): 1390–1403.

    Article  Google Scholar 

  6. BERTRAND H, POLY F, VAN V T, LOMBARD N, NALIN R, VOGEL T M, SIMONET P. High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction [J]. Journal of Microbiological Methods, 2005, 62(1): 1–11.

    Article  Google Scholar 

  7. TSAI Y L, OLSON B H. Rapid method for direct extraction of DNA from soil and sediments [J]. Applied and Environmental Microbiology, 1991, 57(4): 1070–1074.

    Google Scholar 

  8. ZHOU J Z, BRUNS M A, TIEDJE J M. DNA recovery from soils of diverse composition [J]. Applied and Environmental Microbiology, 1996, 62(2): 316–322.

    Google Scholar 

  9. WANG F P, ZHOU H Y, MENG J, PENG X T, JIANG L J, SUN P, ZHANG C L, VAN NOSTRAND J D, DENG Y, HE Z L, WU L Y, ZHOU J H, XIAO X. GeoChip-based analysis of metabolic diversity of microbial communities at the Juan de fuca ridge hydrothermal vent [J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(12): 4840–4845.

    Article  Google Scholar 

  10. ZHOU J Z, KANG S, SCHADT C W, GARTEN C T. Spatial scaling of functional gene diversity across various microbial taxa [J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(22): 7768–7773.

    Article  Google Scholar 

  11. WILSON I G. Inhibition and facilitation of nucleic acid amplification [J]. Applied and Environmental Microbiology, 1997, 63(10): 3741–3751.

    Google Scholar 

  12. JACKSON C R, HARPER J P, WILLOUGHBY D, RODEN E E, CHURCHILL P F. A simple, efficient method for the separation of humic substances and DNA from environmental samples [J]. Applied and Environmental Microbiology, 1997, 63(12): 4993–4995.

    Google Scholar 

  13. PERSOH D, THEUERL S, BUSCOT F, RAMBOLD G. Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil [J]. Journal of Microbiological Methods, 2008, 75(1): 19–24.

    Article  Google Scholar 

  14. CHO J C, LEE D H, CHO Y C, CHO J C, KIM S J. Direct extraction of DNA from soil for amplification of 16S rRNA gene sequences by polymerase chain reaction [J]. Journal of Microbiology, 1996, 34(3): 229–235.

    Google Scholar 

  15. FROSTEGARD A, COURTOIS S, RAMISSE V, CLERC S, BERNILLON D, LE GALL F, JEANNIN P, NESME X, SIMONET P. Quantification of bias related to the extraction of DNA directly from soils [J]. Applied and Environmental Microbiology, 1999, 65(12): 5409–5420.

    Google Scholar 

  16. LEFF L G, DANA J R, MCARTHUR J V, SHIMKETS L J. Comparison of methods of dna extraction from stream sediments [J]. Applied and Environmental Microbiology, 1995, 61(3): 1141–1143.

    Google Scholar 

  17. JACOBSEN C S, RASMUSSEN O F. Development and application of a new method to extract bacterial-DNA from soil based on separation of bacteria from soil with cation-exchange resin [J]. Applied and Environmental Microbiology, 1992, 58(8): 2458–2462.

    Google Scholar 

  18. ROOSE-AMSALEG C L, GARNIER-SILLAM E, HARRY M. Extraction and purification of microbial DNA from soil and sediment samples [J]. Applied Soil Ecology, 2001, 18(1): 47–60.

    Article  Google Scholar 

  19. MILLER D N, BRYANT J E, MADSEN E L, GHIORSE W C. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples [J]. Applied and Environmental Microbiology, 1999, 65(11): 4715–4724.

    Google Scholar 

  20. TECHER D, MARTINEZ-CHOIS C, D’INNOCENZO M, LAVAL-GILLY P, BENNASROUNE A, FOUCAUD L, FALLA J. Novel perspectives to purify genomic DNA from high humic acid content and contaminated soils [J]. Separation and Purification Technology, 2010, 75(1): 81–86.

    Article  Google Scholar 

  21. MOREIRA D. Efficient removal of PCR inhibitors using agarose-embedded DNA preparations [J]. Nucleic Acids Research, 1998, 26(13): 3309–3310.

    Article  Google Scholar 

  22. WIESLANDER L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels [J]. Analytical Biochemistry, 1979, 98(2): 305–309.

    Article  Google Scholar 

  23. WU L Y, LIU X, SCHADT C W, ZHOU J Z. Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification [J]. Applied and Environmental Microbiology, 2006, 72(7): 4931–4941.

    Article  Google Scholar 

  24. HAYES M. Humus chemistry-Genesis, composition, reactions [J]. Nature, 1983, 303(5920): 835–836.

    Article  Google Scholar 

  25. AKANE A, MATSUBARA K, NAKAMURA H, TAKAHASHI S, KIMURA K. Purification of highly degraded DNA by gel-filtration for PCR [J]. Biotechniques, 1994, 16(2): 235–238.

    Google Scholar 

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Authors and Affiliations

  1. School of Minerals Processing and Bioengineering, Central South University, Changsha, 410083, China

    Jian-ping Xie  (谢建平) & Xin-xing Liu  (刘新星)

  2. Institute for Environmental Genomics, Department of Botany and Microbiology, University of Oklahoma, Norman, OK, 73072, USA

    Jian-ping Xie  (谢建平), Li-you Wu  (吴力游), J. D. van Nostrand, Zhi-li He  (贺志理), Zhen-mei Lü  (吕镇梅), Hao Yu  (于浩), Jin-bo Xiong  (熊金波) & Ji-zhong Zhou  (周集中)

  3. Key Laboratory of Biometallurgy of Ministry of Education (Central South University), Changsha, 410083, China

    Jian-ping Xie  (谢建平), Xin-xing Liu  (刘新星) & Ji-zhong Zhou  (周集中)

  4. College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

    Zhen-mei Lü  (吕镇梅) & Ji-zhong Zhou  (周集中)

  5. College of Resource and Environment Engineering, Liaoning Technical University, Fuxin, 123000, China

    Hao Yu  (于浩)

  6. State Key Laboratory of Agricultural Microbiology, (Huazhong Agricultural University), Wuhan, 430070, China

    Jin-bo Xiong  (熊金波)

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  1. Jian-ping Xie  (谢建平)
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  2. Li-you Wu  (吴力游)
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  3. J. D. van Nostrand
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  4. Zhi-li He  (贺志理)
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  6. Hao Yu  (于浩)
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  7. Jin-bo Xiong  (熊金波)
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  8. Xin-xing Liu  (刘新星)
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  9. Ji-zhong Zhou  (周集中)
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Corresponding author

Correspondence to Jian-ping Xie  (谢建平).

Additional information

Foundation item: Project(51104189) supported by the National Natural Science Foundation of China; Project(2010CB630901) supported by the National Basic Research Program of China; Project(1343-77341) supported by the Graduate Education Innovative Program of Central South University, China; Project(DOE-ER64125) supported by Department of Energy, Office of Science under the Environmental Remediation Science Program of the United States

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Xie, Jp., Wu, Ly., van Nostrand, J.D. et al. Improvements on environmental DNA extraction and purification procedures for matagenomic analysis. J. Cent. South Univ. 19, 3055–3063 (2012). https://doi.org/10.1007/s11771-012-1378-6

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  • DOI: https://doi.org/10.1007/s11771-012-1378-6

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