Robust DNA protocols for tropical soils

Author(s)

Venturini, Andressa Monteiro ^[1] ; Nakamura, Fernanda Mancini ^[1] ; Gontijo, Julia Brandao ^[1] ; da Franca, Aline Giovana ^[1] ; Yoshiura, Caio Augusto ^[1] ; Mandro, Jessica Adriele ^[1] ; Tsai, Siu Mui ^[1]

Affiliation

^[1] Univ Sao Paulo, Ctr Nucl Energy Agr, Cell & Mol Biol Lab, Ave Centenario 303, BR-13416000 Piracicaba, SP - Brazil

• Document type: Journal article
• Source: HELIYON; v. 6, n. 5 MAY 2020.
• Web of Science Citations: 2

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 varzea, 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 varzea 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. (AU)

• FAPESP's process: 15/12282-6 - MICROCOSMS AND THE ROLE OF ACTIVE MICROBIOTA ON METHANE CYCLE IN SOILS UNDER FORESTS AND PASTURE OF EASTERN AMAZON
• Grantee: Fernanda Mancini Nakamura
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 15/08564-6 - Molecular bioindicators of soil microbiome from corn crop under straw effect to N2O gas mitigation
• Grantee: Caio Augusto Yoshiura
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 15/13546-7 - Microbial community resilience capacity assessed by metagenomics and metatranscriptomics in Amazon forest and pasture soils
• Grantee: Andressa Monteiro Venturini
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 18/14974-0 - Dynamics of methane and sulfur microbial communities in Amazonian soils
• Grantee: Júlia Brandão Gontijo
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/50320-4 - Dimensions US-BIOTA - São Paulo: collaborative research: integrating dimensions of microbial biodiversity across land use change in tropical forests
• Grantee: Tsai Siu Mui
• Support Opportunities: BIOTA-FAPESP Program - Thematic Grants
• FAPESP's process: 15/23758-1 - Functional genes of the methane biogeochemical cycle in forest and pastures areas in Amazon
• Grantee: Jéssica Adriele Mandro
• Support Opportunities: Scholarships in Brazil - Scientific Initiation