Simple and rapid method for direct extraction of microbial DNA from soil for PCR

• Published in: Soil biology & biochemistry 1998-08, Vol.30 (8-9), p.983-993
• Main Authors: CULLEN, D. W, HIRSCH, P. R
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biochemistry and biology; Biological and medical sciences; Chemical, physicochemical, biochemical and biological properties; Fundamental and applied biological sciences. Psychology; Microbiology; Physics, chemistry, biochemistry and biology of agricultural and forest soils; Rhizobium leguminosarum; Soil science
• ISSN: 0038-0717; 1879-3428
• DOI: 10.1016/S0038-0717(98)00001-7

A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Co-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum bv. viciae strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25 plus or minus 1.7 mu g crude DNA g super(-1) wet soil (i.e. 20 mu g g super(-1) dry soil), sheared to fragment sizes of about 22-25 kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442 bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10 mg super(-1) soil against a background of 10 super(7) diverse non-target bacteria.