Application of Locked Nucleic Acid (LNA) Oligonucleotide–PCR Clamping Technique to Selectively PCR Amplify the SSU rRNA Genes of Bacteria in Investigating the Plant-Associated Community Structures
スポンサーリンク
概要
- 論文の詳細を見る
The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide–PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3'end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide–PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes.
- 日本微生物生態学会の論文
日本微生物生態学会 | 論文
- B-24 PD-PCR : 蛍光消光プローブとプライマーを用いた遺伝子構成比の新規アッセイ方法(モニタリング,(2)口頭発表会,研究発表会)
- PB-24 プラズマ細胞破砕によるDNA均等抽出の検討(群集構造解析,ポスターセッションB,ポスター発表)
- 05-083 バンコマイシン耐性腸球菌に感染するバクテリオファージの分離とその特性(環境衛生,研究発表)
- 11-215 糸状菌細胞内生細菌の検出および系統解析(共生/相互作用,研究発表)
- 11-211 土壌から検出される菌核の微生物担体としての機能(土壌生態系,研究発表)