title: Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria.
abstract: 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.
authors.affiliation: a Faculty of Agriculture , Kagoshima University , Kagoshima , Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ikenaga
authors.forename: Masakazu
authors.initials: M
authors.lastname: Tabuchi
authors.forename: Takuya
authors.initials: T
authors.lastname: Oyama
authors.forename: Isao
authors.initials: I
authors.lastname: Akagi
authors.forename: Masao
authors.initials: M
authors.lastname: Sakai
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D058645:Denaturing Gradient Gel Electrophoresis; D020459:Genes, rRNA; D008928:Mitochondria / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics*; D010944:Plants / Q000235:genetics / Q000382:microbiology; D018087:Plastids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; C477371:locked nucleic acid
keywords: LNA oligonucleotide; PCR clamping; community structure; plant organelle; plant-associated bacteria