Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase.
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 phi29 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 10(5) 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 phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-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.
Yokouchi
Hiroko
H
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Fukuoka
Yayoi
Y
Mukoyama
Daikichi
D
Calugay
Ronie
R
Takeyama
Haruko
H
Matsunaga
Tadashi
T
D000818:Animals; D037421:Anthozoa / Q000382:microbiology*; D017103:Bacillus Phages / Q000201:enzymology; D001431:Bacteriological Techniques / Q000379:methods*; D044822:Biodiversity; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D017753:Ecosystem; D015723:Gene Library; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D004259:DNA-Directed DNA Polymerase