Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis

A reliable method for characterizing microbial communities on the basis of their differences in the 16S ribosomal RNA (rRNA) gene sequences in the hot arid zone sandy soils has been optimized. A desert plant (Calligonum polygonoides) was chosen to provide the rhizospheric soil samples, collected fro...

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Autores principales:	Kumar Gothwal,Raj, Kumar Nigam,Vinod, Mohan,M. Krishna, Sasmal,Dinakar, Ghosh,Purnendu
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2007
Materias:	arid soils microbial community PCR-DGGE rhizosphere soil DNA extraction
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582007000300007
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spelling	oai:scielo:S0717-345820070003000072007-11-05Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysisKumar Gothwal,RajKumar Nigam,VinodMohan,M. KrishnaSasmal,DinakarGhosh,Purnendu arid soils microbial community PCR-DGGE rhizosphere soil DNA extraction A reliable method for characterizing microbial communities on the basis of their differences in the 16S ribosomal RNA (rRNA) gene sequences in the hot arid zone sandy soils has been optimized. A desert plant (Calligonum polygonoides) was chosen to provide the rhizospheric soil samples, collected from three different agro-ecological locations. Total community DNA was efficiently extracted at small-scale level using direct lysis with hot sodium dodecyl sulphate (SDS), glass bead beating and finally subjecting the sandy soil to liquid nitrogen freeze-thaw cycles. To amplify V3 region of bacterial 16S rRNA gene, universal conserved primers were used. Second round polymerase chain reaction (PCR) was attempted to increase product concentration and to minimize the effect of inhibitory substances. To enhance the detection sensitivity of the denaturing gradient gel electrophoresis (DGGE), the effect of change in template DNA concentration was studied. The separation of bands were greatly enhanced in the fingerprints obtained after the second round of PCR representing low abundant species which were not differentiated at single optimized concentration of DNAinfo:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.10 n.3 20072007-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582007000300007en10.4067/S0717-34582007000300007
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	arid soils microbial community PCR-DGGE rhizosphere soil DNA extraction
spellingShingle	arid soils microbial community PCR-DGGE rhizosphere soil DNA extraction Kumar Gothwal,Raj Kumar Nigam,Vinod Mohan,M. Krishna Sasmal,Dinakar Ghosh,Purnendu Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
description	A reliable method for characterizing microbial communities on the basis of their differences in the 16S ribosomal RNA (rRNA) gene sequences in the hot arid zone sandy soils has been optimized. A desert plant (Calligonum polygonoides) was chosen to provide the rhizospheric soil samples, collected from three different agro-ecological locations. Total community DNA was efficiently extracted at small-scale level using direct lysis with hot sodium dodecyl sulphate (SDS), glass bead beating and finally subjecting the sandy soil to liquid nitrogen freeze-thaw cycles. To amplify V3 region of bacterial 16S rRNA gene, universal conserved primers were used. Second round polymerase chain reaction (PCR) was attempted to increase product concentration and to minimize the effect of inhibitory substances. To enhance the detection sensitivity of the denaturing gradient gel electrophoresis (DGGE), the effect of change in template DNA concentration was studied. The separation of bands were greatly enhanced in the fingerprints obtained after the second round of PCR representing low abundant species which were not differentiated at single optimized concentration of DNA
author	Kumar Gothwal,Raj Kumar Nigam,Vinod Mohan,M. Krishna Sasmal,Dinakar Ghosh,Purnendu
author_facet	Kumar Gothwal,Raj Kumar Nigam,Vinod Mohan,M. Krishna Sasmal,Dinakar Ghosh,Purnendu
author_sort	Kumar Gothwal,Raj
title	Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
title_short	Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
title_full	Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
title_fullStr	Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
title_full_unstemmed	Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
title_sort	extraction of bulk dna from thar desert soils for optimization of pcr-dgge based microbial community analysis
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2007
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582007000300007
work_keys_str_mv	AT kumargothwalraj extractionofbulkdnafromthardesertsoilsforoptimizationofpcrdggebasedmicrobialcommunityanalysis AT kumarnigamvinod extractionofbulkdnafromthardesertsoilsforoptimizationofpcrdggebasedmicrobialcommunityanalysis AT mohanmkrishna extractionofbulkdnafromthardesertsoilsforoptimizationofpcrdggebasedmicrobialcommunityanalysis AT sasmaldinakar extractionofbulkdnafromthardesertsoilsforoptimizationofpcrdggebasedmicrobialcommunityanalysis AT ghoshpurnendu extractionofbulkdnafromthardesertsoilsforoptimizationofpcrdggebasedmicrobialcommunityanalysis
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Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis

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