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Methods for microbial DNA extraction from soil for PCR amplification

Abstract

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

References

  1. Yeates, C., Gillings, M. R., Davison, A. D., Altavilla, N. and Veal, D. A. 1997. PCR amplification of crude microbial DNA extracted from soil. Letters in Applied Microbiology 25, 303–307.

    Article  PubMed  CAS  Google Scholar 

  2. Atlas, R. M. 1984. Diversity of microbial communities. Advances in Microbial Ecology 7, 1–47.

    Google Scholar 

  3. Ogram, A., Sayler, G. S. and Barkay, T. 1987. The extraction and purification of microbial DNA from sediments. Journal of Microbiological Methods 7, 57–66.

    Article  CAS  Google Scholar 

  4. Steffan, R. J., Goksoyr, J., Bej, A. K. and Atlas, R. M. 1988. Recovery of DNA from soils and sediments. Applied and Environmental Microbiology 54, 2908–2915.

    PubMed  CAS  Google Scholar 

  5. Holben, W. E., Jansson, J. K., Chelm, B. K. and Tiedje, J. M. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Applied and Environmental Microbiology 54, 703–711.

    PubMed  CAS  Google Scholar 

  6. Porteous, L. A. and Armstrong, J. L. 1991. Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Current Microbiology 22, 345–348.

    Article  CAS  Google Scholar 

  7. Borneman, J., Skroch, P. W., O’Sullivan, K. M., Palus, J. A., Rumjanek, N. G., Jansen, J. L., Nienhuis, J. and Triplett, E. W. 1996. Molecular microbial diversity of an agricultural soil in Wisconsin. Applied and Environmental Microbiology 62, 1935–1943.

    PubMed  CAS  Google Scholar 

  8. More, M. I., Herrick, J. B., Silva, M. C., Ghiorse, W. C. and Madsen, E. L. 1994. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Applied and Environmental Microbiology 60, 1572–1580.

    PubMed  CAS  Google Scholar 

  9. Tebbe, C. C. and Vahjen, W. 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Applied and Environmental Microbiology 59, 2657–2665.

    PubMed  CAS  Google Scholar 

  10. Tsai, Y.-L. and Olson, B. H. 1991. Rapid method for direct extraction of DNA from soil and sediments. Applied and Environmental Microbiology 57, 1070–1074.

    PubMed  CAS  Google Scholar 

  11. Zhou, J., Bruns, M. A. and Tiedje, J. M. 1996. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology 62, 316–322.

    PubMed  CAS  Google Scholar 

  12. Sambrook, J., Fritsch, E. F. and Maniatis, T.. 1987. Molecular cloning — a laboratory manual. Cold Spring Harbor, USA, Cold Spring Harbor Laboratory Press.

    Google Scholar 

  13. Picard, C., Ponsonnet, C., Paget, E., Nesme, X. and Simonet, P. 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722.

    PubMed  CAS  Google Scholar 

  14. Steffan, R. J. and Atlas, R. M. 1988. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Applied and Environmental Microbiology 54, 2185–2191.

    PubMed  CAS  Google Scholar 

  15. Liesack, W., Weyland, H. and Stackebrandt, E. 1991. Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria. Microbial Ecology 21, 191–198.

    Article  CAS  Google Scholar 

  16. Tsai, Y.-L. and Olson, B. H. 1992. Detection of low numbers of bacterial numbers in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757.

    PubMed  CAS  Google Scholar 

  17. Holben, W. E. 1994. Isolation and purification of bacterial DNA from soil. Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. Madison, USA, Soil Science Society of America: 727–751.

  18. Tsai, Y.-L. and Olson, B. H. 1992. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Applied and Environmental Microbiology 58, 2292–2295.

    PubMed  CAS  Google Scholar 

  19. Edwards, U., Rogall, T., Blocker, H., Emde, M. and Bottger, E. C. 1989. Isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research 17, 7843–7853.

    Article  PubMed  CAS  Google Scholar 

  20. Jensen, M. A., Webster, J. A. and Straus, N. 1993. Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Applied and Environmental Microbiology 59, 945–952.

    PubMed  CAS  Google Scholar 

  21. White, T. J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of ribosomal RNA genes for phylogenetics. PCR protocols : a guide to methods and applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, Academic Press: 315–322.

  22. Gillings, M. R. 1998. Personal communication.

  23. Yap, W. H., Li, X., Soong, T. W. and Davies, J. E. 1996. Genetic diversity of soil microorganisms assessed by analysis of hsp70 (dnaK) sequences. Journal of Industrial Microbiology 17, 179–184.

    Article  CAS  Google Scholar 

  24. Koenraadt, H., Somerville, S. C. and Jones, A. L. 1992. Characterisation of mutations in the betatubulin gene of benomyl resistant field strains of Venturia inaequalis and other pathogenic fungi. Phytopathology 82, 1348–1354.

    Article  Google Scholar 

  25. Zehr, J. P. and McReynolds, L. A. 1989. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Applied and Environmental Microbiology 55, 2522–2526.

    PubMed  CAS  Google Scholar 

  26. Porteous, L. A., Armstrong, J. L., Seidler, R. J. and Watrud, L. S. 1994. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. Current Microbiology 29, 301–307.

    Article  PubMed  CAS  Google Scholar 

  27. Volossiouk, T., Robb, E. J. and Nazar, R. N. 1995. Direct DNA extraction for PCR-mediated assays of soil organisms. Applied and Environmental Microbiology 61, 3972–3976.

    PubMed  CAS  Google Scholar 

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Yeates, C., Gillings, M.R., Davison, A.D. et al. Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1, 40–47 (1998). https://doi.org/10.1251/bpo6

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