Top

Research Article

Split Viewer

Mol. Cells 2012; 34(4): 383-391

Published online October 31, 2012

https://doi.org/10.1007/s10059-012-0169-0

© The Korean Society for Molecular and Cellular Biology

Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

Jae-Ho Jung1,7, Sanghee Kim2,7, Seongho Ryu3,7, Min-Seok Kim1, Ye-Seul Baek1, Se-Joo Kim1,6, Joong- Ki Choi4, Joong-Ki Park5, and Gi-Sik Min1,*

1Department of Biological Sciences, Inha University, Incheon 402-751, Korea, 2Korea Polar Research Institute (KORDI), Songdo Techno Park, Incheon 406-840, Korea, 3Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA, 4Department of Oceanography, Inha University, Incheon 402-751, Korea, 5Department of Parasitology and Graduate Program in Cell Biol-ogy and Genetics, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea, 6Present address: Deep-sea and Seabed Resources Research Division, Korea Institute of Ocean Science and Technology, Ansan 426-744, Korea, 7These authors contrib-uted equally to this work.

Correspondence to : *Correspondence: mingisik@inha.ac.kr

Received: June 26, 2012; Revised: July 26, 2012; Accepted: July 27, 2012

Abstract

Despite recent advance in mass sequencing technolo-gies such as pyrosequencing, assessment of culture-indepen-dent 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 target-ing a particular group would provide enhanced sensitiv-ity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific 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.

Keywords ciliate, community analysis, phylum-specific PCR, pyrosequencing, SNP, SSU rRNA

Article

Research Article

Mol. Cells 2012; 34(4): 383-391

Published online October 31, 2012 https://doi.org/10.1007/s10059-012-0169-0

Copyright © The Korean Society for Molecular and Cellular Biology.

Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

Jae-Ho Jung1,7, Sanghee Kim2,7, Seongho Ryu3,7, Min-Seok Kim1, Ye-Seul Baek1, Se-Joo Kim1,6, Joong- Ki Choi4, Joong-Ki Park5, and Gi-Sik Min1,*

1Department of Biological Sciences, Inha University, Incheon 402-751, Korea, 2Korea Polar Research Institute (KORDI), Songdo Techno Park, Incheon 406-840, Korea, 3Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA, 4Department of Oceanography, Inha University, Incheon 402-751, Korea, 5Department of Parasitology and Graduate Program in Cell Biol-ogy and Genetics, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea, 6Present address: Deep-sea and Seabed Resources Research Division, Korea Institute of Ocean Science and Technology, Ansan 426-744, Korea, 7These authors contrib-uted equally to this work.

Correspondence to:*Correspondence: mingisik@inha.ac.kr

Received: June 26, 2012; Revised: July 26, 2012; Accepted: July 27, 2012

Abstract

Despite recent advance in mass sequencing technolo-gies such as pyrosequencing, assessment of culture-indepen-dent 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 target-ing a particular group would provide enhanced sensitiv-ity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific 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.

Keywords: ciliate, community analysis, phylum-specific PCR, pyrosequencing, SNP, SSU rRNA

Mol. Cells
Sep 30, 2022 Vol.45 No.9, pp. 603~672
COVER PICTURE
The Target of Rapamycin Complex (TORC) is a central regulatory hub in eukaryotes, which is well conserved in diverse plant species, including tomato (Solanum lycopersicum). Inhibition of TORC genes (SlTOR, SlLST8, and SlRAPTOR) by VIGS (virus-induced gene silencing) results in early fruit ripening in tomato. The red/ orange tomatoes are early-ripened TORC-silenced fruits, while the green tomato is a control fruit. Top, left, control fruit (TRV2-myc); top, right, TRV2-SlLST8; bottom, left, TRV2-SlTOR; bottom, right, TRV2-SlRAPTOR(Choi et al., pp. 660-672).

Supplementary File

Share this article on

  • line
  • mail

Related articles in Mol. Cells

Molecules and Cells

eISSN 0219-1032
qr-code Download