Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community
| Autor: | Lucy Seldin, Jan Dirk van Elsas, Alexandre S. Rosado, A.C. Keijzer-Wolters, Gabriela Frois Duarte |
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| Jazyk: | angličtina |
| Rok vydání: | 1998 |
| Předmět: |
Microbiology (medical)
Genetics Instituut voor Plantenziektenkundig Onderzoek Denaturing gradient gel electrophoresis Research Institute for Plant Protection Ribosomal RNA Biology Microbiology law.invention chemistry.chemical_compound genomic DNA Soil chemistry Bacterial diversity law Large ribosomal subunit Internal transcribed spacer Molecular Biology Ribosomal DNA DNA Temperature gradient gel electrophoresis Polymerase chain reaction |
| Zdroj: | Journal of Microbiological Methods, 32(1), 21-29 Journal of Microbiological Methods 32 (1998) 1 |
| ISSN: | 0167-7012 |
| DOI: | 10.1016/s0167-7012(98)00004-9 |
| Popis: | A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils of different texture. DNA and rRNA yields from these soils were 15–30 and 0.25–1.0 μg g −1 soil, respectively. Following different purification steps, the rRNA as well as genomic DNA extracts obtained were sufficiently pure for either reverse transcription and polymerase chain reaction (PCR) amplification, or direct PCR amplification. Using a set of universal bacterial primers based on conserved regions of the 16S rRNA sequence, both approaches yielded mixed target molecules for subsequent denaturing gradient gel electrophoresis fingerprinting of soil microbial diversity. The amplified rRNA-based bacterial diversity assessment was compared with diversity assessments based on amplified DNA in one selected soil. Results showed similarities as well as differences between the profiles generated on the basis of rRNA and those based on genomic DNA, which suggested that the bacterial communities defined on the basis of their genomic DNA contained variable amounts of rRNA. |
| Databáze: | OpenAIRE |
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