An improved method compatible with metagenomic analyses to extract genomic DNA from soils in Tuber melanosporum orchards
Autor: | Pascale Frey-Klett, F. Le Tacon, Stéphane Uroz, Aurélie Deveau, Sanjay Antony-Babu, Claude Murat |
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Přispěvatelé: | Interactions Arbres-Microorganismes (IAM), Université de Lorraine (UL)-Institut National de la Recherche Agronomique (INRA), Université de Lorraine (UL), Centre des Sciences du Goût et de l'Alimentation [Dijon] (CSGA), Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique (CNRS), French National Research Agency [ANR-09-STRA-10], INRA (Institut National de la Recherche Agronomique) Center of Nancy, French Research Agency through the Laboratory of Excellence ARBRE [ANR-12-LABXARBRE-01], Institut National de la Recherche Agronomique (INRA)-Université de Lorraine (UL), Centre National de la Recherche Scientifique (CNRS)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB), ANR-09-STRA-0010,SYSTRUF,Bases d'une gestion écologique durable, des écosystèmes truffiers (producteurs de Tuber melanosporum)(2009), ANR-11-LABX-0002,ARBRE,Recherches Avancées sur l'Arbre et les Ecosytèmes Forestiers(2011), ANR: ANR-12-LABXARBRE-01, Interactions Arbres-Microorganismes ( IAM ), Institut National de la Recherche Agronomique ( INRA ) -Université de Lorraine ( UL ), Université de Lorraine ( UL ), Centre des Sciences du Goût et de l'Alimentation [Dijon] ( CSGA ), Institut National de la Recherche Agronomique ( INRA ) -Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique ( CNRS ) |
Jazyk: | angličtina |
Rok vydání: | 2013 |
Předmět: |
DNA
Bacterial Tuber melanosporum GENES [SDV]Life Sciences [q-bio] DIVERSITY Biology Applied Microbiology and Biotechnology soil BLACK-TRUFFLE MECHANISMS 03 medical and health sciences FOREST SOIL Ascomycota calcareous soil MICROARRAY Botany STRATEGY DNA extraction Soil Microbiology 030304 developmental biology 2. Zero hunger guanidine thiocyanate 0303 health sciences Truffle PURIFICATION Bacteria [ SDV ] Life Sciences [q-bio] Denaturing Gradient Gel Electrophoresis MICROBIAL COMMUNITY EDTA DNA 04 agricultural and veterinary sciences General Medicine 15. Life on land QUANTITY genomic DNA Horticulture [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology Metagenomics 040103 agronomy & agriculture 0401 agriculture forestry and fisheries Pyrosequencing 260:230 nm ratio Soil microbiology Temperature gradient gel electrophoresis Biotechnology |
Zdroj: | Journal of Applied Microbiology Journal of Applied Microbiology, Wiley, 2013, 115 (1), pp.163-170. ⟨10.1111/jam.12205⟩ Journal of Applied Microbiology, Wiley, 2013, 115 (1), pp.163-170. 〈10.1111/jam.12205〉 |
ISSN: | 1364-5072 1365-2672 |
DOI: | 10.1111/jam.12205⟩ |
Popis: | 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 (gDNA). The work presented here aims to present an inherent problem with 260 : 230 nm ratio of extracted gDNA from calcareous soils of Tuber melanosporum orchards and a protocol to overcome this problem. Methods and Results Using two commercial gDNA extraction kits on spatially distant truffle orchards, we demonstrated that the 260 : 230 nm ratio was very low, consequentially yielding gDNA 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 gDNA 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 gDNA from calcareous soils of T. melanosporum orchards. Significance and Impact of the Study The method recommended here significantly improves gDNA quality obtained from T. melanosporum orchards to make it acceptable for highly sensitive methods such as microarray. |
Databáze: | OpenAIRE |
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