Abstract
Microorganism DNA of rhizosphere soil from Pinus koraiensis and Pinus sylvestriformis were extracted by proteinase K based on SDS method, CTAB method, PVP (polyvinylpolypyrrolidone) method, and freezing and thawing method and the crude DNA from rhizosphere soil were purified by dialysis method, silver beads absorption method, and squeezing DNA gel method. The results of different extracting and purifying methods were compared and evaluated. Results indicated that the best method of extraction for microorganism DNA in rhizosphere soil was proteinse K based on SDS method with high salt concentration of 1.0% (w/v) NaCl, which could effectively eliminate humic acids and other impurities. The dialysis method was suitable to purify DNA from rhizosphere soil because of effectively removing brown matters and humic acids and the purified products were suited to PCR amplification. Squeezing DNA gel method was also a good purification method with the advantage of inexpensive in cost and efficient in use.
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References
Amann, R.I., Ludwig, W., Schleifer, K.H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation [J]. Microbiol. Rev., 59: 143–169.
Brock, T.D. 1987. The study of microorganisms in situ: progress and problems [J], Soc. Gene Microbiol., 41: 1–17.
Chen Han, Tang Xiaoshu, Lin Jie, Zhang Bosheng, Ren Daming. 2002. Community constitute and phylogenetic analysis on soil uncultured microorganism [J]. Acta. Microbiol. Sinica, 42: 478–483. (in Chinese)
Courtois, S., Frostegard, A.S., Goransson, P., et al. 2001. Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or fromcells previously released by density gradient centrifugation [J]. Environ. Micribiol., 7: 431–439.
Ellinsøe, P., Johnsen, K. 2002. Influence of soil sample sizes on the assessment of bacterial community structure [J]. Soil Biolo. & Biochmi., 34: 1701–1707.
Emilio, O., Casamayor, PedrÓs-AliÓ, C., Muyzer, G., et al. 2002. Microheterogeneity in 16S ribosomal DNA — defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations [J]. Appl. and Environ. Microbiol., 68: 1706–1717.
England, L.S., Trevors, J.T., Holmes, S.B. 2001. Extraction and detection of baculoviral DNA from lake water, detritus and forest litter [J]. J of Appl. Microbiol., 90: 630–636.
Fujimoto, C., Maeda, H., Kokeguchi, S., et al. 2003. Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingginal plaque [J]. J Pediodont Res., 38: 440–445.
Haws worth, D.L. 1991. The fungal dimension of biodiversity magnitude, Significance, and conservation [J]. Logical Res., 95: 641–655.
Head, I.M., Saunders, J.R., Pickup, R.W. 1998. Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms [J]. Microbial Ecol., 35: 1–21.
Laurent, M.F., Philippot, L., Hallet, S., et al. 2001. DNA extraction from soils: Old bias for new microbial diversity analysis methods [J]. Appl. & environ. Microbiol., 67: 2354–2359.
Liesack, W., Stackrandt, E. 1992. Occurrence of novel groups of the domain bacterial as revealed by analysis of genetic material isolated from an Australian terrestrial environment [J]. J Bacteriol., 174: 5072–5078.
Luo Haifeng, Qi Hongyan, Xue Kai, Wang Xiaoyi, Wang Chuan, Zhang Hongxun. 2003. Influence of application of GC-clamp on study of soil microbial diversity by PCR-DGGE [J]. Acta Econlogica. Sinica., 23(10): 2170–2175. (in Chinese)
Luo Haifeng, Qi Hongyan, Xue Kai, Zhang Hongxun. 2003. A preliminary application of PCR-DGGE to study microbial diversity in soil [J]. Acta Ecologica Sinica, 23(8): 1570–1575. (in Chinese)
Ma Yuexin, Holmström, C., Webb, J., et al. 2003. Application of denaturing gradient el electrophoresis (DGGE) in microbial ecology [J]. Acta Ecologica Sinica., 23(8): 1561–1569. (in Chinese)
Ogram, A., Sayler, G.S., Barkay, T. 1987. The extraction and purification of microbial DNA from sediments [J]. J. Microbiol. Methods, 7: 7–66.
Paterson, E. 2003. Importance of rhizodeposition in the coupling of plant andmicro-bial productivity [J]. European J of Soil Sci., 10: 1–10.
Ranjard, L., David, P.H., Lejon, et al. 2003. Sampling strategy in molecular microbial ecology: influence of fungal and bacterial communities [J]. Environ. Microbiol., 11: 1111–1120.
Schmalenberger, A., Christoph, C., Tebbe. 2003. Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies [J]. Molecular Ecol. 12: 251–262.
Smalla, K., Cresswell, N., Hagler, L.C.M., et al. 1993. Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification [J]. J. Appl. Bacteriol., 74: 78–85.
Tesar, M., Thomas, G., Reichenauer, et al. 2002. Bacterial rhizosphere populations of black popular and herbal plants to be used for phytoremediation of diesel fule [J]. Soil Biology & Biochem., 34: 1883–1892.
Tsai, Y.L., Betty, H.O. 1996. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction [J]. Appl. Environ. Microbiol., 58: 2292–2295.
Ward, D.M., Weller, R., Basteston, M.M. 1990. rDNA sequences reveal numerous uncultured inhabitants in a natural community [J]. Nature, 345: 63–65.
Zhang Ruifu, Cao Hui, Cui Zhongli, Li Shunpeng, Fan Ben. 2003. Extraction and purification of soil microbial total DNA [J]. Acta. Microbiol. Sini., 43(2): 276–282. (in Chinese)
Zhou, J.Z., Bruns, M., James, M., et al. 1996. DNA recovery from soils of diverse composition [J]. Appl. and Environ. Microbiol., 62: 316–322.
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Foundation item: This project was supported by the Knowledge Innovation Project from Chinese Academy (KZCX1-SW-01) and the National Natural Science Foundation of China (30070158).
Biography: JIA Xia (1975–), female, Ph. D. work in Environmental Science and Engineering College, Chang’an University, Xi’an 710054, P. R. China.
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Jia, X., Han, Sj., Zhao, Yh. et al. Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil. J. of For. Res. 17, 31–34 (2006). https://doi.org/10.1007/s11676-006-0007-3
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DOI: https://doi.org/10.1007/s11676-006-0007-3