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An improved method compatible with metagenomic analyses to extract genomic DNA from soils in T uber melanosporum orchards
Author(s) -
AntonyBabu S.,
Murat C.,
Deveau A.,
Tacon F.,
FreyKlett P.,
Uroz S.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12205
Subject(s) - genomic dna , dna extraction , biology , extraction (chemistry) , dna microarray , metagenomics , pyrosequencing , microbiology and biotechnology , chromatography , polymerase chain reaction , dna , chemistry , genetics , gene , gene expression
Abstract Aims The development of high‐throughput methods such as pyrosequencing and microarrays has greatly improved our understanding of the microbial diversity in complex environments such as soils. Nevertheless, albeit advancements in such techniques, the first major step is to obtain high quantity and good quality genomic DNA (g DNA ). The work presented here aims to present an inherent problem with 260 : 230 nm ratio of extracted g DNA from calcareous soils of T uber melanosporum orchards and a protocol to overcome this problem. Methods and Results Using two commercial g DNA extraction kits on spatially distant truffle orchards, we demonstrated that the 260 : 230 nm ratio was very low, consequentially yielding g DNA incompatible with microarray analyses. In order to solve this problem, optimization steps were tested including several wash steps performed before and/or after lysis. These washes significantly improved the g DNA quality (ratio 260 : 230 nm >1·7) without modification of the structure of the bacterial communities as stated by temporal temperature gradient gel electrophoresis analysis. A final re‐extraction with phenol/chloroform was required for one of the soil samples. Conclusions A combination of wash steps included into the extraction protocol followed by phenol: chloroform re‐extraction is recommended to obtain high‐quality g DNA from calcareous soils of T . melanosporum orchards. Significance and Impact of the Study The method recommended here significantly improves g DNA quality obtained from T . melanosporum orchards to make it acceptable for highly sensitive methods such as microarray.