Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of the swrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodiumdodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L.) expressing the StSy gene for resveratrol production and the bar gene for Basta�� tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genus Bacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.
DNA extraction from soil: comparison of different methods using spore-forming bacteria and the swrAA gene as indicators
BALESTRAZZI, ALMA;BONADEI, MARTINA;CALVIO, CINZIA;GALIZZI, ALESSANDRO;CARBONERA, DANIELA
2009-01-01
Abstract
Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of the swrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodiumdodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L.) expressing the StSy gene for resveratrol production and the bar gene for Basta�� tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genus Bacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
