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Current Microbiology

, Volume 29, Issue 5, pp 301–307 | Cite as

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

  • L. Arlene Porteous
  • John L. Armstrong
  • Ramon J. Seidler
  • Lidia S. Watrud
Article

Abstract

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 μg DNA/g (wet weight) of soil and 400 μg DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample “fingerprints.” Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Keywords

Bacillus Pseudomonas Streptomyces Environmental Sample Isothiocyanate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Literature Cited

  1. 1.
    Baker SS, Rugh CL, Kamalay JC (1990) RNA and DNA isolation from recalcitrant plant tissues. Bio Techniques 9:268–272Google Scholar
  2. 2.
    Bej AK, Steffan RJ, DiCesare J, Haff L, Atlas RM (1990) Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl Environ Microbiol 56:307–314Google Scholar
  3. 3.
    Chaudhry GR, Toranzos GA, Ghatti AR (1989) Novel method for monitoring genetically engineered microorganisms in the environment. Appl Environ Microbiol 55:1301–1304Google Scholar
  4. 4.
    Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21Google Scholar
  5. 5.
    Doyle JJ, Dickson EE (1987) Preservation of plant samples for DNA restriction endonuclease analysis. Taxon 36:715–722Google Scholar
  6. 6.
    Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  7. 7.
    Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349Google Scholar
  8. 8.
    Giovannoni SJ, Britschgi TB, Moyer CL, Field KG (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 344:60–63Google Scholar
  9. 9.
    Hilger AB, Myrold DD (1991) Method for extraction ofFrankia DNA from soil. Agric Ecosyst & Environ 34:107–113Google Scholar
  10. 10.
    Liesack W, Stackebrandt E (1992) Occurrence of novel groups of the domainBacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol 174:5072–5078Google Scholar
  11. 11.
    Lippke JA, Strzempko MN, Raia FF, Simon SL, French CK (1987) Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate. Appl Environ Microbiol 53:2588–2589Google Scholar
  12. 12.
    Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325Google Scholar
  13. 13.
    Neefs J-M, Van de Peer Y, Hendriks L, De Wachter R (1990) Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 18:2237–2330Google Scholar
  14. 14.
    Picard C, Ponsonnet C, Paget E, Nesme X, Simonet P (1992) Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol 58:2717–2722Google Scholar
  15. 15.
    Pillai SD, Josephson KL, Bailey RL, Gerba CP, Pepper IL (1991) Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Appl Environ Microbiol 57:2283–2286Google Scholar
  16. 16.
    Porteous LA, Armstrong JL (1991) Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Curr Microbiol 22:345–348Google Scholar
  17. 17.
    Porteous LA, Armstrong JL (1993) A simple mini-method to extract DNA directly from soil for use with polymerase chain reaction amplification. Curr Microbiol 27:115–118Google Scholar
  18. 18.
    Rochelle PA, Olson BH (1991) A simple technique for electroelution of DNA from environmental samples. Bio Techniques 11:724–728Google Scholar
  19. 19.
    Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76Google Scholar
  20. 20.
    Schmidt TM, DeLong EF, Pace NR (1991) Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol 173:4371–4378Google Scholar
  21. 21.
    Steffan RJ, Goksøyr J, Bej AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915Google Scholar
  22. 22.
    Steffan RJ, Atlas RM (1991) Polymerase chain reaction: applications in environmental microbiology. Annu Rev Microbiol 45:137–161Google Scholar
  23. 23.
    Stevenson IL (1958) The effect of sonic vibration on the bacterial plate count of soil. Plant Soil 1:1–8Google Scholar
  24. 24.
    Torsvik V, Goksøyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782–787Google Scholar
  25. 25.
    Tsai Y-L, Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57:1070–1074Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1994

Authors and Affiliations

  • L. Arlene Porteous
    • 1
  • John L. Armstrong
    • 1
  • Ramon J. Seidler
    • 1
  • Lidia S. Watrud
    • 1
  1. 1.Biotechnology ProgramUnited States Environmental Protection Agency, Environmental Research LaboratoryCorvallisUSA

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