Extraction of bulk DNA from Thar Desert soils for optimization of PCR-DGGE based microbial community analysis
Electron. j. biotechnol
; 10(3): 400-408, July 2007. ilus, tab
Artigo
em Inglês
| LILACS
| ID: lil-640485
Biblioteca responsável:
CL1.1
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) 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.
Texto completo:
Disponível
Coleções:
Bases de dados internacionais
Base de dados:
LILACS
Idioma:
Inglês
Revista:
Electron. j. biotechnol
Assunto da revista:
Biotecnologia
Ano de publicação:
2007
Tipo de documento:
Artigo
País de afiliação:
Índia
Instituição/País de afiliação:
Birla Institute of Scientific Research/IN