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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 10, No. 3, 2007, pp. 400-408
Bioline Code: ej07038
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 10, No. 3, 2007, pp. 400-408

 en Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
Gothwal, Raj Kumar; Nigam, Vinod Kumar; Mohan, M. Krishna; Sasmal, Dinakar & Ghosh, Purnendu

Abstract

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 check for this species in other resources ) 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.

Keywords
arid soils, microbial community, PCR-DGGE, rhizosphere, soil DNA extraction.

 
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