Quantification of bacterial subgroups in soil: comparison of DNA extracteddirectly from soil or from cells previously released by density gradient centrifugation

All molecular analyses of soil bacterial diversity are based on the extract ion of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are fir st removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which c ells were first separated from the soil matrix by Nycodenz gradient centrif ugation in order to evaluate the effect of these different approaches on th e analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRN A gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA templat e and amount of PCR product and the influence of competing DNA on PCR ampli fication, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 mug g(-1) dry weight soil versus 38-93 mug g(-1) obtained by in situ lysis methods). However, with the exception of the gamma -subclass of Proteobacteria, there was no significant difference in the spectrum of div ersity resulting from the two extraction strategies.