Molecules and Cells

, Volume 34, Issue 4, pp 383–391 | Cite as

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


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.


ciliate community analysis phylum-specific PCR pyrosequencing SNP SSU rRNA 


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Copyright information

© The Korean Society for Molecular and Cellular Biology and Springer Netherlands 2012

Authors and Affiliations

  • Jae-Ho Jung
    • 1
  • Sanghee Kim
    • 2
  • Seongho Ryu
    • 3
  • Min-Seok Kim
    • 1
  • Ye-Seul Baek
    • 1
  • Se-Joo Kim
    • 1
    • 6
  • Joong-Ki Choi
    • 4
  • Joong-Ki Park
    • 5
  • Gi-Sik Min
    • 1
  1. 1.Department of Biological SciencesInha UniversityIncheonKorea
  2. 2.Korea Polar Research Institute (KORDI)IncheonKorea
  3. 3.Department of Cell and Developmental BiologyWeill Cornell Medical CollegeNew YorkUSA
  4. 4.Department of OceanographyInha UniversityIncheonKorea
  5. 5.Department of Parasitology and Graduate Program in Cell Biology and Genetics, College of MedicineChungbuk National UniversityCheongjuKorea
  6. 6.Deep-sea and Seabed Resources Research DivisionKorea Institute of Ocean Science and TechnologyAnsanKorea

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