DNA extraction from soils: old bias for new microbial diversity analysis methods

Abstract

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

FIG. 1

ARDRA. Shown is a SYBR green II-stained gel (6% acrylamide) of RsaI-digested PCR products amplified with 16S rDNA universal primers (27_f and 1492_r) from DNA extracted from Dijon, Couhins, and Epoisses soils following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right).

FIG. 2

RISA shown is a SYBR green II-stained gel (6% acryamide) of PCR products amplified with 16S rDNA intergenic spacer universal primers (38_r and 72_f) from DNA extracted from Dijon, Couhins, and Epoisses soils following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). Arrow, 800-bp band.

FIG. 3

ARDRA. Shown is a SYBR green II-stained gel (6% acrylamide) of RsaI-digested PCR products amplified with 16S rDNA universal primers (27_f and 1492_r) from DNA extracted from unamended soil (U) and farmyard manure (FM)- and sewage sludge (SS10 and SS100)-treated plots of Couhins following three different extraction methods: the MS laboratory method (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). SS10, 10 tons of dry matter/ha/years. SS100, 100 tons of dry matter/ha/2 years.

FIG. 4

RISA. Shown is SYBR green II-stained gel (6% acryamide) of PCR products amplified with 16S ribosomal universal primers (38_r and 72_f) from DNA extracted from unamended soil (U) and farmyard manure (FM)- or sewage sludge (SS10 and SS100)-treated plots of Couhins soil following three different extraction methods: the MS laboratory method (MS) (lanes 1 to 3), the MoBio kit method (lanes 4 to 6), and the Bio 101 kit method (lanes 7 to 9). Lanes M, VIII Boehringer Mannheim molecular size markers (sizes indicated in base pairs at left and right). Arrow, 800-bp band.