Rapid method for purification of soil DNA for hybridization and PCR analysis.
Monitoring of microbial DNA in soils by dot blot hybridization and PCR analysis is a useful technique for gaining insight into the survival and impact of genetically modified micro-organisms released in the environment. Most methods of DNA isolation from soils require a large number of purification steps rendering them unsuitable for quantitative analysis of multiple samples. Here we describe a very rapid method for the isolation and purification of multiple samples of soil DNA that can be used directly for dot blot hybridization and PCR analysis. Soil DNA extracts are prepared by lysozyme/SDS treatment at pH 9.0 and purified by ammonium acetate precipitation and Sephadex G50 gel filtration. In a practical application of this method, sandy soil samples were seeded with Alcaligenes eutrophus cells and exposed to high temperature (42 degrees C) or desiccation. As a result, the number of culturable A. eutrophus cells which could be recovered from the soil samples quickly declined. However, the concentration of a marker gene encoding resistance to cadmium, cobalt and zinc (czc) remained unaltered.
Dijkmans
R
R
Laboratory of Genetics and Biotechnology, Vlaamse Instelling voor Technologisch Onderzoek (VITO), Mol, Belgium.
Jagers
A
A
Kreps
S
S
Collard
J M
JM
Mergeay
M
M
D000421:Alcaligenes / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012988:Soil Microbiology
D004269:DNA, Bacterial