Robust DNA protocols for tropical soils

Andressa Monteiro Venturini; Fernanda Mancini Nakamura; Júlia Brandão Gontijo; Aline Giovana da França; Caio Augusto Yoshiura; Jéssica Adriele Mandro; Siu Mui Tsai

doi:10.1016/j.heliyon.2020.e03830

Robust DNA protocols for tropical soils

Heliyon. 2020 May 13;6(5):e03830. doi: 10.1016/j.heliyon.2020.e03830. eCollection 2020 May.

Authors

Andressa Monteiro Venturini¹, Fernanda Mancini Nakamura¹, Júlia Brandão Gontijo¹, Aline Giovana da França¹, Caio Augusto Yoshiura¹, Jéssica Adriele Mandro¹, Siu Mui Tsai¹

Affiliation

¹ Cell and Molecular Biology Laboratory, Center for Nuclear Energy in Agriculture, University of São Paulo, Avenida Centenário, 303, Piracicaba, SP, 13416-000, Brazil.

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 várzea, 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 várzea 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.

Keywords: Amazonian soils; Archaeal communities; Bacteria; Bacterial communities; Biodiversity; DNA extraction; Microbial ecology; Microbial genomics; Microbiology; Microorganism; Molecular biology; Polymerase chain reaction; Quantitative real-time PCR.; Soil biology; Soil science.