Quantification of bias related to the extraction of DNA directly from soils

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed fo r the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an or ganism inoculated into the soil. The aim of the present investigation was t o improve the extraction, purification, and quantification of DNA derived f rom as large a portion of the soil microbial community as possible, with sp ecial emphasis placed on obtaining DNA from gram-positive bacteria, which f orm structures that are difficult to disrupt. Furthermore, we wanted to ide ntify and minimize the biases related to each step in the procedure. Six so ils, covering a range of pHs, clay contents, and organic matter contents, w ere studied. Lysis was carried out by soil grinding, sonication, thermal sh ocks, and chemical treatments. DNA was extracted from the indigenous microf lora as well as from inoculated bacterial cells, spores, and hyphae, and th e quality and quantity of the DNA were determined by gel electrophoresis an d dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield com pared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay c ontent strongly influenced the recovery of DNA. The adsorption of DNA to cl ay particles decreased when the soil was pretreated with RNA in order to sa turate the adsorption sites. We also investigated different purification te chniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging .