Quantification of bias related to the extraction of DNA directly from soils

Abstract

In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage lambda DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

FIG. 1

Protocols showing successive treatments for direct in situ lysis of soil microorganisms for DNA extraction. For details, see Materials and Methods. Protocol 1 consists of extraction of extracellular DNA from dried soil, with no lysis treatment. Protocol 2 includes grinding of dried soil followed by DNA extraction. Protocol 3 includes soil grinding followed by homogenization in the extraction buffer using an Ultraturrax mixer. In protocols 4a and b, soil grinding and homogenization are followed by sonication using a titanium microtip sonicator (protocol 4a) or a Cup Horn sonicator (protocol 4b). Protocols 5a and b include soil grinding, homogenization, and sonication using a microtip (protocol 5a) or a Cup Horn (protocol 5b) sonicator, followed by chemical and enzymatic lysis.

FIG. 2

Gel electrophoresis (0.8% agarose) of DNA extracted from 300 mg of soil 3 (Côte St André) after different lysis treatments (protocols 1 to 5 [see Fig. 1]). Lanes M, lambda DNA size marker.

FIG. 3

Proportion of actinomycetes belonging to different genera in relation to the total viable actinomycetes after lysis treatments 1 through 5 (see Fig. 1). The number of CFU was determined on a medium selecting for this group of bacteria. A total of about 400 colonies were examined.

FIG. 4

Recovery of HindIII-digested phage lambda DNA added to soils at different concentrations before (G) or after (G∗) grinding. T (thermal shocks) and S (sonication) are additional lysis treatments. Quantification was carried out by phosphorimaging after dot blot hybridization. For each soil, one sample was used for each concentration of phage lambda DNA added. Soil characteristics are described in Table 1. The samples corresponding to 10 and 15 μg of added DNA, soil 4, were lost during preparation.

FIG. 5

PCR amplification of DNA extracted from soil 3 according to lysis protocols 1, 2, 3, 5a, and 5b. Primers FGPS122 and FGPS350 (Table 2) were used to target indigenous Streptosporangium spp. The DNA extract either was undiluted or was diluted 10- or 100-fold. Lanes M, 123-bp molecular marker (Gibco BRL); lanes C, control without DNA. Arrow indicates the 513-bp amplification product.

FIG. 6

Amounts of DNA recovered from S. lividans OS48.3 spores (a) and mycelium (b) inoculated into soils at different concentrations. The quantities of mycelium added to the soil corresponded to the number of spores inoculated into the germination medium. Of these, about 50% germinated. The exact number of cells or genomes in the hyphae of the germinated spores was not determined. Therefore, the amounts of inoculated spores and mycelium are not directly comparable. Extraction was carried out by lysis protocol 6 (see Materials and Methods). A prime (′) indicates that RNA was included in the extraction buffer. Target DNA was PCR amplified with primers FGPS516 and FGPS517. Quantification was done by phosphorimaging after dot blot hybridization using the oligonucleotide probe FFPS518. For each soil, one sample was used for each concentration of hyphae or spores. Soil characteristics are described in Table 1.