SHORT COMMUNICATION Experimental discrimination and molecular characterization of the extracellular soil DNA fraction Maria Teresa Ceccherini Æ Judith Ascher Æ Alberto Agnelli Æ Federica Borgogni Æ Ottorino Luca Pantani Æ Giacomo Pietramellara Received: 12 March 2009 / Accepted: 2 June 2009 / Published online: 17 June 2009 Ó Springer Science+Business Media B.V. 2009 Abstract We experimentally discriminated and quali-quantitatively characterized the extracellular fraction of a forest soil DNA pool. We sequentially extracted and classified the components of extracel- lular DNA by its strength of interaction with soil colloids as: (1) extractable in water, free in the extracellular soil environment or adsorbed on soil colloids; and as (2) extractable in alkaline buffer after previous extraction in water, bound on soil colloids. The comparative molecular analysis (fluorometer, gel electrophoresis, genetic fingerprinting) of directly and sequentially extracted extracellular DNA revealed quantitative and qualitative differences, also in terms of genetic information about microbial communities. The sequential extraction of extracel- lular DNA revealed differences in molecular weight, indicating a relationship between DNA fragment length and strength of interaction with soil colloids. The sequential extraction was also suitable to assess the presence of tightly bound DNA, providing information about the DNA-colloid interactions nat- urally occurring in the soil environment. Keywords DGGE Á Extracellular soil DNA (eDNA) Á eDNA components Á Microbial communities The total soil DNA pool (tDNA) consists of an intracellular (iDNA) and an extracellular (eDNA) fraction. Soil eDNA is quantitatively relevant, with a high survival capacity and mobility, playing a crucial role in the gene transfer by transformation, in the formation of bacterial biofilm and as a source of nutrients for soil microorganisms (Agnelli et al. 2004, 2007; Ceccherini et al. 2007; Levy-Booth et al. 2007; Ascher et al. 2009a; Pietramellara et al. 2009). Concerning the discrimination of the environmental DNA pool, a few studies have separately considered the eDNA and iDNA fraction of sediments (Ogram et al. 1987; dell’Anno et al. 2002; Corinaldesi et al. 2005). The available literature on the adsorption and binding of DNA on soil colloids like clay and sand minerals, humic substances, Fe–Al oxides/hydroxides and their complexes (Pietramellara et al. 2009) evidences, that DNA molecules can interact with these substrates with different strength, resulting in differently mobile components of the eDNA fraction. In genetic fingerprinting studies of soil microbial communities, eDNA has rarely been considered due M. T. Ceccherini Á J. Ascher (&) Á F. Borgogni Á O. L. Pantani Á G. Pietramellara Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Universita ` degli Studi di Firenze, Piazzale delle Cascine 28, 50144 Florence, Italy e-mail: judith.ascher@unifi.it A. Agnelli Dipartimento di Scienze Agrarie ed Ambientali, Universita ` degli Studi di Perugia, Borgo XX Giugno, 06121 Perugia, Italy 123 Antonie van Leeuwenhoek (2009) 96:653–657 DOI 10.1007/s10482-009-9354-3