Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using φ29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 105 cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using φ29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Bata Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that α-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.
ASJC Scopus subject areas
- Ecology, Evolution, Behavior and Systematics