Robust DNA protocols for tropical soils

Abstract

Studies in the Amazon are being intensified to evaluate the alterations in the microbial communities of soils and sediments in the face of increasing deforestation and land-use changes in the region. However, since these environments present highly heterogeneous physicochemical properties, including contaminants that hinder nucleic acids isolation and downstream techniques, the development of best molecular practices is crucial. This work aimed to optimize standard protocols for DNA extraction and gene quantification by quantitative real-time PCR (qPCR) based on natural and anthropogenic soils and sediments (primary forest, pasture, Amazonian Dark Earth, and v arzea, a seasonally flooded area) of the Eastern Amazon. Our modified extraction protocol increased the fluorometric DNA concentration by 48%, reaching twice the original amount for most of the pasture and v arzea samples, and the 260/280 purity ratio by 15% to values between 1.8 to 2.0, considered ideal for DNA. The addition of bovine serum albumin in the qPCR reaction improved the quantification of the 16S rRNA genes of Archaea and Bacteria and its precision among technical replicates, as well as allowed their detection in previously non-amplifiable samples. It is concluded that the changes made in the protocols improved the parameters of the DNA samples and their amplification, thus increasing the reliability of microbial communities' analysis and its ecological interpretations.

Genome-resolved metagenomics reveals novel archaeal and bacterial genomes from Amazonian forest and pasture soils

“…Forest and pasture soil samples under original soil moisture and at 100 % field capacity were DNA-extracted in duplicate using the PowerLyzer PowerSoil DNA Isolation Kit (QIAGEN, Hilden, North Rhine-Westphalia, Germany), totaling 12 DNA samples, following a protocol optimized for Amazon soils [22]. DNA samples were checked using 1 % agarose gel electrophoresis and a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and stored at −20 °C.…”

Increased soil moisture intensifies the impacts of forest-to-pasture conversion on methane emissions and methane-cycling communities in the Eastern Amazon

“…Total DNA was extracted from soil samples collected on day 30 (T30) from all treatments. Extractions were carried out in duplicate from 0.25 g of soil each using the PowerLyzer PowerSoil DNA Isolation Kit (QIA-GEN, Hilden, North Rhine Westphalia, Germany), following a modified version of the manufacturer's protocol optimized for Amazon soils (Venturini et al, 2020): at the initial stage, after adding the solution C1, the samples were vortexed for 15 min at maximum speed and centrifuged for 3 min at 10,000×g; and following the addition of the solutions C2 and C3, the incubations were made at − 20 • C instead of 4 • C. The concentration and purity of the DNA samples were evaluated by 1% agarose gel electrophoresis and using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The DNA samples were stored at − 20 • C until processed.…”

Maintaining grass coverage increases methane uptake in Amazonian pastures, with a reduction of methanogenic archaea in the rhizosphere

“…DNA was extracted for molecular analyses from soils used in greenhouse experiments, previously originated from Ariquemes region and from soils of the field study in the Tapajós region. Total DNA was extracted from soil samples using the PowerLyzer PowerSoil DNA Isolation Kit (Qiagen, Hilden, Germany) from 250 mg of soil, according to the protocol provided by the manufacturer, except that after adding solution C1 the stirring time was extended to 15 min followed by 3 min centrifugation (Venturini et al, 2020). The amount and quality of the DNA extracted were analyzed in a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an optical density of 260 nm.…”