Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

Abstract

All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 microg g(-1) dry weight soil versus 38-93 microg g(-1) obtained by in situ lysis methods). However, with the exception of the gamma-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.

Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

“…Because DNA survives for some time in soils following cell death (11), it is possible that the higher estimates observed using 16S rRNA gene targets may in part be attributed to the presence of dead cells. Methods to extract live bacterial cells from soils have been developed to try to overcome this potential bias (1,14), but such approaches also suffer from inherent bias (28), and 100% extraction efficiency is unlikely. Nevertheless, the same trend of a reduction in rhizobia was observed in this data set, as has been consistently observed using the MPN approach.…”

Extraction of DNA from soil

“…By using hybridization of the PCR amplified 16S rDNA gene Courtois et al [10] did not find significant difference in the spectrum of diversity resulting from the two extraction strategies, the only exception being the gamma-subclass of Proteobacteria.…”

“…Even when evaluation is attempted, the positive or negative effects of extraction parameters is difficult to extrapolate to other samples. However, some comparative studies have contributed to better understanding the value of extraction and/or purification steps when applied to previously characterized soil samples [10,17,25,33,42,77].…”

“…The second approach separates the cell fraction from soil particles first. Then, this bacterial fraction is lysed and the nucleic acids purified [10,23,67]. Both approaches have advantages and disadvantages related to DNA yields, DNA purity for molecular purposes, and the unbiased representation of the entire microbial diversity [10,58].…”

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

“…A single fecal sample from an individual was obtained from a collaborator (Dr. Martin Blaser, NYU), stored at À80°C, and preprocessed prior to flow sorting using a Nycodenz purification procedure (Courtois et al 2001). Briefly, ;1 g of fecal material was placed in 10 mL of 13 PBS with 3-mm glass beads in a 50-mL conical centrifuge and vigorously vortexed for 3 min.…”