A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities

A single DNA procedure to recover bacterial DNA from various soil microenvi ronments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (ou tside and surface of aggregates), the inner part or microporosity (inside o f aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and ce ll disruption by grinding in liquid nitrogen, followed by enzymatic lysis w ith lysozyme and proteinase K. High yields of high molecular weight DNA (gr eater than or equal to 23 kb) were obtained for all microenvironments. Crud e DNA yields for the various soil microenvironments were between 0.7 and 51 .4 mu g DNA.g(-1) soil sample and were positively correlated with bacterial cell abundance (r = 0.91). Further purification steps allowed to recover a t least 60 % of the DNA extracted from the Various microenvironments. The s uitability of the extracted DNA to undergo enzymatic amplification reaction s and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacteria l 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (be ta- and gamma-proteobacteria, High G+C Gram-positive bacteria, and Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation (nifH) and mercu ry resistance (merRT Delta P) genes. (C) Elsevier, Paris.