Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria

• Published in: Bioscience, biotechnology, and biochemistry Vol. 79; no. 9; pp. 1556 - 1566
• Main Authors: Ikenaga, Makoto, Tabuchi, Masakazu, Oyama, Takuya, Akagi, Isao, Sakai, Masao
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 02.09.2015; Oxford University Press
• Subjects: Bacteria - genetics; Bacteria - isolation & purification; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; community structure; Denaturing Gradient Gel Electrophoresis; Genes, rRNA; LNA oligonucleotide; Mitochondria - genetics; Oligonucleotides - genetics; PCR clamping; plant organelle; plant-associated bacteria; Plants - genetics; Plants - microbiology; Plastids - genetics; Polymerase Chain Reaction - methods; PCR clamping; LNA oligonucleotide; community structure; plant organelle; plant-associated bacteria
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1080/09168451.2015.1038213

Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria. DGGE patterns of SSU rRNA genes derived from soybean and potato samples. Symbols "-" and "+" indicated the lanes generated without (0 μM) and with (4.0 μM) LNA oligonucleotides.