Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis

Abstract

The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding-freezing-thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel electrophoresis. This indicates the importance of the DNA extraction protocol on data for evaluating soil bacterial diversity. Consistently, the bead-beating procedure gave rise to the highest number of DNA bands, indicating the highest number of bacterial species. Supplementing the bead-beating procedure with additional cell-rupture steps generally did not change the bacterial community profile. The same consistency was not observed when evaluating the efficiency of the different methods on soil DNA yield. This parameter depended on soil type. The DNA size was of highest molecular weight with the sonication and grinding-freezing-thawing procedures (approx. 20 kb). In contrast, the inclusion of bead beating resulted in more sheared DNA (approx. 6-20 kb), and the longer the bead-beating time, the higher the fraction of low-molecular weight DNA. Clearly, the choice of DNA extraction protocol depends on soil type. We found, however, that for the analysis of indigenous soil bacterial communities the bead-beating procedure was appropriate because it is fast, reproducible, and gives very pure DNA of relatively high molecular weight. And very importantly, with this protocol the highest soil bacterial diversity was obtained. We believe that the choice of DNA extraction protocol will influence not only the determined phylogenetic diversity of indigenous microbial communities, but also the obtained functional diversity. This means that the detected presence of a functional gene - and thus the indication of enzyme activity - may depend on the nature of the applied DNA extraction procedure. © 2004 Elsevier Ltd. All rights reserved.