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

Microbes Environ. 2018 Sep 29;33(3):340-344. doi: 10.1264/jsme2.ME18071. Epub 2018 Aug 24.

Abstract

PCR clamping by locked nucleic acid (LNA) oligonucleotides is an effective technique for selectively amplifying the community SSU rRNA genes of plant-associated bacteria. However, the original primer set often shows low amplification efficiency. In order to improve this efficiency, new primers were designed at positions to compete with LNA oligonucleotides. Three new sets displayed higher amplification efficiencies than the original; however, efficiency varied among the primer sets. Two new sets appeared to be available in consideration of bacterial profiles by next-generation sequencing. One new set, KU63f and KU1494r, may be applicable to the selective gene amplification of plant-associated bacteria.

Keywords: KU63f and KU1494r; LNA oligonucleotide; PCR clamping; SSU rRNA genes; plant–associated bacteria.

Publication types

  • Evaluation Study

MeSH terms

  • Bacteria / classification
  • Bacteria / genetics*
  • Bacteria / isolation & purification
  • Bacterial Typing Techniques / methods*
  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • High-Throughput Nucleotide Sequencing
  • Microbiota / genetics*
  • Mitochondria / genetics
  • Oligonucleotides / genetics*
  • Plants / genetics
  • Plants / microbiology*
  • Plastids / genetics
  • Polymerase Chain Reaction*
  • Ribosome Subunits, Small, Bacterial / genetics*
  • Sequence Analysis, DNA

Substances

  • DNA Primers
  • DNA, Bacterial
  • Oligonucleotides
  • locked nucleic acid