Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Nov;61(11):3972–3976. doi: 10.1128/aem.61.11.3972-3976.1995

Direct DNA extraction for PCR-mediated assays of soil organisms.

T Volossiouk 1, E J Robb 1, R N Nazar 1
PMCID: PMC167704  PMID: 8526511

Abstract

By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  2. Haqqi T. M., Sarkar G., David C. S., Sommer S. S. Specific amplification with PCR of a refractory segment of genomic DNA. Nucleic Acids Res. 1988 Dec 23;16(24):11844–11844. doi: 10.1093/nar/16.24.11844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Holben William E., Jansson Janet K., Chelm Barry K., Tiedje James M. DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community. Appl Environ Microbiol. 1988 Mar;54(3):703–711. doi: 10.1128/aem.54.3.703-711.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  5. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  6. Picard C., Ponsonnet C., Paget E., Nesme X., Simonet P. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol. 1992 Sep;58(9):2717–2722. doi: 10.1128/aem.58.9.2717-2722.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Pillai S. D., Josephson K. L., Bailey R. L., Gerba C. P., Pepper I. L. Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Appl Environ Microbiol. 1991 Aug;57(8):2283–2286. doi: 10.1128/aem.57.8.2283-2286.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. STEELE W. J., OKAMURA N., BUSCH H. EFFECTS OF THIOACETAMIDE ON THE COMPOSITION AND BIOSYNTHESIS OF NUCLEOLAR AND NUCLEAR RIBONUCLEIC ACID IN RAT LIVER. J Biol Chem. 1965 Apr;240:1742–1749. [PubMed] [Google Scholar]
  10. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  11. Steffan R. J., Atlas R. M. Polymerase chain reaction: applications in environmental microbiology. Annu Rev Microbiol. 1991;45:137–161. doi: 10.1146/annurev.mi.45.100191.001033. [DOI] [PubMed] [Google Scholar]
  12. Steffan R. J., Goksøyr J., Bej A. K., Atlas R. M. Recovery of DNA from soils and sediments. Appl Environ Microbiol. 1988 Dec;54(12):2908–2915. doi: 10.1128/aem.54.12.2908-2915.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES