TY - JOUR
T1 - Limitations and benefits of ARISA intra-genomic diversity fingerprinting
AU - Popa, Radu
AU - Popa, Rodica
AU - Mashall, Matthew J.
AU - Nguyen, Hien
AU - Tebo, Bradley M.
AU - Brauer, Suzanna
N1 - Funding Information:
This work was supported by a research grant from a NASA Astrobiology Program (NNH07ZDA001N-EXOB) and by a 2008 Portland State University Faculty Enhancement Grant. We thank Dr. Mitch Cruzan and Trieste Dobberstein (PSU) for support with using the ABI 310 sequencer, Dr. Niles Lehman (PSU) for access to the Typhoon gel reader, Dr. James K. Fredrickson (PNNL) for Shewanella strains from the PNNL culture collection, Dr. Ken Stedman for access to the gradient PCR instrument and Dr. Kenneth Nealson, Dr. Ana Obratsova and Shana Rapoport for strains from the USC culture collection.
PY - 2009/8
Y1 - 2009/8
N2 - Monitoring diversity changes and contamination in mixed cultures and simple microcosms is challenged by fast community structure dynamics, and the need for means allowing fast, cost-efficient and accurate identification of microorganisms at high phylogenetic resolution. The method we explored is a variant of Automated rRNA Intergenic Spacer Analysis based on Intra-Genomic Diversity Fingerprinting (ARISA-IGDF), and identifies phylotypes with multiple 16S-23S rRNA gene Intergenic Transcribed Spacers. We verified the effect of PCR conditions (annealing temperature, duration of final extension, number of cycles, group-specific primers and formamide) on ARISA-IGD fingerprints of 44 strains of Shewanella. We present a digitization algorithm and data analysis procedures needed to determine confidence in strain identification. Though using stringent PCR conditions and group-specific primers allow reasonably accurate identification of strains with three ARISA-IGD amplicons within the 82-1000 bp size range, ARISA-IGDF is best for phylotypes with ≥ 4 unambiguously different amplicons. This method allows monitoring the occurrence of culturable microbes and can be implemented in applications requiring high phylogenetic resolution, reproducibility, low cost and high throughput such as identifying contamination and monitoring the evolution of diversity in mixed cultures and low diversity microcosms and periodic screening of small microbial culture libraries.
AB - Monitoring diversity changes and contamination in mixed cultures and simple microcosms is challenged by fast community structure dynamics, and the need for means allowing fast, cost-efficient and accurate identification of microorganisms at high phylogenetic resolution. The method we explored is a variant of Automated rRNA Intergenic Spacer Analysis based on Intra-Genomic Diversity Fingerprinting (ARISA-IGDF), and identifies phylotypes with multiple 16S-23S rRNA gene Intergenic Transcribed Spacers. We verified the effect of PCR conditions (annealing temperature, duration of final extension, number of cycles, group-specific primers and formamide) on ARISA-IGD fingerprints of 44 strains of Shewanella. We present a digitization algorithm and data analysis procedures needed to determine confidence in strain identification. Though using stringent PCR conditions and group-specific primers allow reasonably accurate identification of strains with three ARISA-IGD amplicons within the 82-1000 bp size range, ARISA-IGDF is best for phylotypes with ≥ 4 unambiguously different amplicons. This method allows monitoring the occurrence of culturable microbes and can be implemented in applications requiring high phylogenetic resolution, reproducibility, low cost and high throughput such as identifying contamination and monitoring the evolution of diversity in mixed cultures and low diversity microcosms and periodic screening of small microbial culture libraries.
KW - ARISA-IGDF
KW - ITS
KW - Molecular fingerprinting
KW - qPCR
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U2 - 10.1016/j.mimet.2009.06.005
DO - 10.1016/j.mimet.2009.06.005
M3 - Review article
C2 - 19538993
AN - SCOPUS:67650104286
SN - 0167-7012
VL - 78
SP - 111
EP - 118
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 2
ER -