Applied Microbiology and Biotechnology

, Volume 64, Issue 5, pp 665–670

DNA isolation from soil samples for cloning in different hosts

Original Paper

Abstract

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.

References

  1. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169Google Scholar
  2. Berthelet M, Whyte LG, Greer CW (1996) Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns. FEMS Microbiol Lett 138:17–22Google Scholar
  3. Borneman J, Skroch PW, O’Sullivan KM, Palus JA, Rumjanek NG, Jansen JL, Nienhuis J, Triplett EW (1996) Molecular microbial diversity of an agricultural soil in Wisconsin. Appl Environ Microbiol 62:1935–1943Google Scholar
  4. Degrange V, Bardin R (1995) Detection and counting of Nitrobacter populations in soil by PCR. Appl Environ Microbiol 61:2093–2098Google Scholar
  5. Entcheva P, Liebl W, Johann A, Hartsch T, Streit WR (2001) Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl Environ Microbiol 67:89–99Google Scholar
  6. Höss M, Pääbo S (1993) DNA extraction from pleistocene bones by a silica-based purification method. Nucleic Acids Res 21:3913–3914Google Scholar
  7. Jacobsen CS, Rasmussen OF (1992) Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation exchange resin. Appl Environ Microbiol 58:2458–2462Google Scholar
  8. Johnston CG, Aust SD (1994) Detection of Phanerochaete chrysosporium in soil by PCR and restriction enzyme analysis. Appl Environ Microbiol 60:2350–2354Google Scholar
  9. La Montagne MG, Michel FC, Holden PA, Reddy CA (2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods 49:255–264Google Scholar
  10. Lee SY, Bollinger J, Bezdicek D, Ogram A (1996) Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method. Appl Environ Microbiol 62:3787–3793Google Scholar
  11. Martin-Laurent F, Philippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001) DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67:2354–2359Google Scholar
  12. Moré MI, Herrick JB, Silva MC, Ghiorse WC, Madsen EL (1994) Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl Environ Microbiol 60:1572–1580Google Scholar
  13. 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
  14. Porteous LA, Armstrong JL, Seidler RJ, Watrud LS (1994) An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. Curr Microbiol 29:301–307Google Scholar
  15. Purdy KJ, Embley TM, Takii S, Nedwell DB (1996) Rapid extraction of DNA and rRNA from sediments by a novel hydroxyapatite spin-column method. Appl Environ Microbiol 62Google Scholar
  16. Roller C, Ludwig W, Schleifer KH (1992) Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes. J Gen Microbiol 138:1167–1175Google Scholar
  17. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  18. Selenska S, Klingmüller W (1991) DNA recovery and direct detection of tn5 sequences from soil. Lett Appl Microbiol 13:21–24Google Scholar
  19. Steffan RJ, Goksoyr J, Bej AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915Google Scholar
  20. Strohl WR (1992) Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res 20:961–974Google Scholar
  21. Tebbe CC, 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. Appl Environ Microbiol 59:2657–2665Google Scholar
  22. Torsvik V, Goksoyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782–787Google Scholar
  23. Tsai YL, Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57:1070–1074Google Scholar
  24. Tsai YL, Olson BH (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol 58:2292–2295Google Scholar
  25. Volossiouk T, Robb EJ, Nazar RN (1995) Direct DNA extraction for PCR-mediated assays of soil organisms. Appl Environ Microbiol 61:3972–3976Google Scholar
  26. Wikstrom P, Wiklund A, Andersson AC, Forsman M (1996) DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types. J Biotechnol 52:107–120Google Scholar
  27. Wintzingerode F von, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229Google Scholar
  28. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Institut für technische BiochemieUniversität StuttgartStuttgartGermany

Personalised recommendations