GENETIC DIVERSITY ANALYSIS OF THERMOPHILIC BACTERIA FROM CANDRADIMUKA CRATER IN CENTRAL JAVA EMPLOYING PCR-RFLP OF 16S-rRNA GENE
AbstractThe specific primers for bacteria (63f and 1387r) were used to amplify the 16S-rRNA genes from total community genomic DNA of thermophilic bacteria. The total community genomic DNA was obtained from muds and water samples of Candradimuka crater, Dieng Plateau, Central Java. PCR products were cloned into vectorÂ pCR*2.1-TOPO (3.9 kb) and transformed into Escherichia coli TOPIC. TwoÂ tetrameric restriction endonucleasesÂ RsalÂ andÂ HhalÂ were employed to generate Restriction Fragment Length Polymorphisms (RFLP) paterns. These enzymes yielded 10 and 9 groups of 16S-rRNA profiles or OTU (Operational Taxonomic Units) from 27 16S-rRNA gene clones. Rsal was found to be more discriminative in differentiating the clones than Hhal. Rsal-RFLP indicated that OTU 7 and OTU 3 represented the most abundant clones, i.e. 6 and 5 clones respectively. The distribution of 16S-rRNA gene clones couldÂ indicate relative distribution ofÂ specific groups ofÂ thermophilicÂ bacteriaÂ inÂ theirÂ natural habitat. Analysis of diversity at the DNA level could represent both culturableÂ andÂ unculturable bacteria in theÂ environment. Similarity analysis showed thatÂ at levelÂ 0.600 thereÂ were 8 differentÂ groups from 10Â RFLPÂ profiles generated byÂ RsalÂ digestion. This study indicated that there were at least 8 groups of different thermophilic bacteria occupying Candradimuka crater. Key words: Thermophiles, 16S-rRNA, Candradimuka crater.
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