A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities
A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (ou...
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Published in | European journal of soil biology Vol. 34; no. 2; pp. 89 - 97 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Paris
Elsevier Masson SAS
01.04.1998
Editions scientifiques et médicales Elsevier Elsevier |
Subjects | |
Online Access | Get full text |
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Abstract | A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g
−1 soil sample and were positively correlated with bacterial cell abundance (
r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-proteobacteria, High G+C Gram-positive bacteria, and
Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation (
nifH) and mercury resistance (
merRTΔP) genes.
Une procédure présentant une efficacité comparable pour extraire directement l'ADN des microenvironnements du sol, différents par leurs caractéristiques physico-chimiques et structurales, a été développée. Ces microenvironnements, obtenus par une technique de lavages de sol combiné à un fractionnement physique, sont le compartiment externe ou macroporosité (surface des agrégats), le compartiment interne ou microporisté (l'intérieur des agrégats) et les différentes classes de taille et de stabilité d'agrégats. La technique d'extraction d'ADN consiste en une homogénéisation de l'échantillon et une lyse physique des cellules par cryobroyage dans l'azote liquide, suivie d'une lyse enzymatique avec du lysozyme et de la proteinase K. Des rendements importants de récupération d'ADN de haute masse moléculaire (≥ 23 kb) ont été obtenu dans chaque microenvironnement. Ces rendements varient de 0,7 à 51,4 μg d'ADN non purifié par gramme de microenvironnement et sont positivement corrélés avec l'abondance en cellule bactérienne (
r = 0,91). L'étape de purification développée à permis de récupérer au moins 60 % de l'ADN extrait pour chaque microenvironnement. La qualité de l'ADN pour effectuer des amplifications par PCR ainsi que l'efficacité de la technique d'extraction pour récupérer l'ADN de différents types cellulaires ont été démontrées par l'amplification de séquences d'ADN ribosomiques spécifiques de différents groupes procaryotes. |
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AbstractList | A single DNA procedure to recover bacterial DNA from various soil microenvironments which differ in their physical, chemical and structural properties was developed. These microenvironments, obtained by a combination of soil washes and physical fractionation, were the outer part or macroporosity (outside and surface of aggregates), the inner part or microporosity (inside of aggregates) and various size and stability classes of soil aggregates and particles. The DNA extraction method involved sample homogenization and cell disruption by grinding in liquid nitrogen, followed by enzymatic lysis with lysozyme and proteinase K. High yields of high molecular weight DNA (≥ 23 kb) were obtained for all microenvironments. Crude DNA yields for the various soil microenvironments were between 0.7 and 51.4 μg DNA·g
−1 soil sample and were positively correlated with bacterial cell abundance (
r = 0.91). Further purification steps allowed to recover at least 60 % of the DNA extracted from the various microenvironments. The suitability of the extracted DNA to undergo enzymatic amplification reactions and the effectiveness of the extraction procedure in recovering DNA from various native bacterial groups was tested using primers for archaebacterial 16S rDNAs, universal and group-specific eubacterial 16S rDNAs primers (β- and γ-proteobacteria, High G+C Gram-positive bacteria, and
Bacillus species and relatives). Successful amplification of less ubiquitous genes was also obtained with primers targeting nitrogen fixation (
nifH) and mercury resistance (
merRTΔP) genes.
Une procédure présentant une efficacité comparable pour extraire directement l'ADN des microenvironnements du sol, différents par leurs caractéristiques physico-chimiques et structurales, a été développée. Ces microenvironnements, obtenus par une technique de lavages de sol combiné à un fractionnement physique, sont le compartiment externe ou macroporosité (surface des agrégats), le compartiment interne ou microporisté (l'intérieur des agrégats) et les différentes classes de taille et de stabilité d'agrégats. La technique d'extraction d'ADN consiste en une homogénéisation de l'échantillon et une lyse physique des cellules par cryobroyage dans l'azote liquide, suivie d'une lyse enzymatique avec du lysozyme et de la proteinase K. Des rendements importants de récupération d'ADN de haute masse moléculaire (≥ 23 kb) ont été obtenu dans chaque microenvironnement. Ces rendements varient de 0,7 à 51,4 μg d'ADN non purifié par gramme de microenvironnement et sont positivement corrélés avec l'abondance en cellule bactérienne (
r = 0,91). L'étape de purification développée à permis de récupérer au moins 60 % de l'ADN extrait pour chaque microenvironnement. La qualité de l'ADN pour effectuer des amplifications par PCR ainsi que l'efficacité de la technique d'extraction pour récupérer l'ADN de différents types cellulaires ont été démontrées par l'amplification de séquences d'ADN ribosomiques spécifiques de différents groupes procaryotes. |
Author | Poly, Franck Combrisson, Jerome Ranjard, Lionel Richaume, Agnès Nazaret, Sylvie |
Author_xml | – sequence: 1 givenname: Lionel surname: Ranjard fullname: Ranjard, Lionel – sequence: 2 givenname: Franck surname: Poly fullname: Poly, Franck – sequence: 3 givenname: Jerome surname: Combrisson fullname: Combrisson, Jerome – sequence: 4 givenname: Agnès surname: Richaume fullname: Richaume, Agnès – sequence: 5 givenname: Sylvie surname: Nazaret fullname: Nazaret, Sylvie email: nazaret@biomserv.univ-lyon1.fr |
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Copyright | 1998 Elsevier, Paris 1999 INIST-CNRS Distributed under a Creative Commons Attribution 4.0 International License |
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DocumentTitleAlternate | Une procédure unique pour l'extraction d'ADN de la surface ou de l'intérieur d'agrégats de différentes classes par tailles permettant l'amplification par PCR des communautés bactériennes |
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Keywords | PCR detection Bacterial DNA distribution bactérienne microenvironnements du sol bacterial distribution détection par PCR soil microenvironments ADN bactérien Polymerase chain reaction Biochemical analysis Silt loam soil Extraction process Cultivated soil Ribosomal DNA Lysis Bacteria Aggregate Microbial community Molecular microenvironment BIOLOGIE MOLECULAIRE |
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SubjectTerms | ADN bactérien Agronomy. Soil science and plant productions bacterial distribution Bacterial DNA Biochemistry and biology Biological and medical sciences Chemical, physicochemical, biochemical and biological properties distribution bactérienne détection par PCR Environmental Sciences Fundamental and applied biological sciences. Psychology Life Sciences Microbiology microenvironnements du sol PCR detection Physics, chemistry, biochemistry and biology of agricultural and forest soils soil microenvironments Soil science |
Title | A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities |
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