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
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