Efficient recovery of environmental DNA for expression cloning by indirect extraction methods

Abstract

Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability for expression cloning in Escherichia coli. Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used ( 6 8% of eukaryotic DNA by indirect methods versus 61^93% by direct lysis protocols). Considering the higher purity, i.e. higher cloning efficiency of DNA isolated by indirect methods, similar numbers of clones carrying prokaryotic inserts could be produced by either strategy. Gene banks prepared from directly extracted DNA, however, are expected to contain large portions of clones with eukaryotic inserts, whereas those constructed from indirectly isolated DNA should mainly contain inserts of bacterial origin. As eukaryotic genetic information is generally not expressed in bacterial host organisms but increases the library size, our findings suggest that the use of indirect DNA isolation methods allows the construction of environmental gene banks of superior quality.

Biogeography of Rhizobium radiobacter and distribution of associated temperate phages in deep subseafloor sediments

“…DNA extraction from sediments DNA extraction was performed according to a protocol described by Lueders et al (2004) and Gabor et al (2003). Briefly, 0.2 ml of B1:1 mixed 0.1 mm and 0.5 mm silica beads and 700 ml PTNbuffer (120 mM NaPO 4 , 125 mM TRIS, 25 mM NaCl, pH 8) were added to 0.5 cm 3 sediment.…”

The effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods

“…Martin-Laurent et al 2001;Luna et al 2006); however, other studies did not identify such a relationship (e.g. Gabor et al 2003).…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…In the indirect extraction method, the microbial cells are separated before cell lysis and DNA extraction and purification, resulting in less non-bacterial DNA (Osborn and Smith 2005). Comparative benchmarking of direct and indirect extraction methods showed that the indirect method yielded 10-to 100-fold lower amounts of DNA than did direct procedures, but revealed a much higher bacterial diversity (Gabor, de Vries et al 2003). The disadvantages of indirect methods are the longer processing time and lower DNA yields.…”