Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism

Jae Ho Jung, Sanghee Kim, Seongho Ryu, Min Seok Kim, Ye Seul Baek, Se Joo Kim, Joong Ki Choi, Joong Ki Park, Gi Sik Min

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or groupspecific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

Original languageEnglish
Pages (from-to)383-391
Number of pages9
JournalMolecules and Cells
Volume34
Issue number4
DOIs
StatePublished - Oct 2012

Keywords

  • Ciliate
  • Community analysis
  • Phylum-specific PCR
  • Pyrosequencing
  • SNP
  • SSU rRNA

Fingerprint

Dive into the research topics of 'Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism'. Together they form a unique fingerprint.

Cite this