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RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM prot...

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Autores principales: Pinaev, Alexander G., Kichko, Arina A., Aksenova, Tatiana S., Safronova, Vera I., Kozhenkova, Elena V., Andronov, Evgeny E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9783177/
https://www.ncbi.nlm.nih.gov/pubmed/36548141
http://dx.doi.org/10.3390/mps5060099
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author Pinaev, Alexander G.
Kichko, Arina A.
Aksenova, Tatiana S.
Safronova, Vera I.
Kozhenkova, Elena V.
Andronov, Evgeny E.
author_facet Pinaev, Alexander G.
Kichko, Arina A.
Aksenova, Tatiana S.
Safronova, Vera I.
Kozhenkova, Elena V.
Andronov, Evgeny E.
author_sort Pinaev, Alexander G.
collection PubMed
description A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption–desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.
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spelling pubmed-97831772022-12-24 RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances Pinaev, Alexander G. Kichko, Arina A. Aksenova, Tatiana S. Safronova, Vera I. Kozhenkova, Elena V. Andronov, Evgeny E. Methods Protoc Protocol A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption–desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required. MDPI 2022-12-16 /pmc/articles/PMC9783177/ /pubmed/36548141 http://dx.doi.org/10.3390/mps5060099 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Pinaev, Alexander G.
Kichko, Arina A.
Aksenova, Tatiana S.
Safronova, Vera I.
Kozhenkova, Elena V.
Andronov, Evgeny E.
RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title_full RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title_fullStr RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title_full_unstemmed RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title_short RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances
title_sort riam: a universal accessible protocol for the isolation of high purity dna from various soils and other humic substances
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9783177/
https://www.ncbi.nlm.nih.gov/pubmed/36548141
http://dx.doi.org/10.3390/mps5060099
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