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.

Improvements in Bacterial Primers to Enhance Selective SSU rRNA Gene Amplification of Plant-associated Bacteria by Applying the LNA Oligonucleotide-PCR Clamping Technique

“…To improve amplification efficiency, three new forward primers and one new reverse primer were designed at positions to compete with LNA oligonucleotides specific for plant organelle (mitochondria and plastid) SSU rRNA genes. The LNA oligonucleotides used herein were described in our previous studies ( 13 , 14 ), and their sequences and properties were shown in supplementary Table S2 . The amplification efficiencies of the newly designed primer sets were compared for their respective PCR products prepared from the roots of rice, potato, soybean, and turnip green.…”

“…2) were cultivated in potted soil. Cultivation procedures were the same as those described by Ikenaga et al ( 13 , 14 ). The roots of rice and soybean were collected at the seedling stage, while those of potato and turnip green were collected at the harvest stage.…”

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

“…We have developed an LNA oligonucleotide–PCR clamping technique in order to investigate the community structures of plant–associated bacteria ( 20 , 21 , 22 ). This technique has enabled the PCR amplification of bacterial SSU rRNA genes, while inhibiting the amplification of host plant organelle (mitochondria and plastid) genes.…”