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

Abstract

A single DNA procedure to recover bacterial DNA from various soil microenvironments 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 (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g −1 soil sample and were positively correlated with bacterial cell abundance ( r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-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 mercury resistance ( merRTΔP) genes.