Limitations and benefits of ARISA intra-genomic diversity fingerprinting.
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 >or=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.
Popa
Radu
R
Department of Biology, Portland State University, 1719 SW 10th Ave., SB2 Room 246, Portland, OR 97201, USA. rpopa@pdx.edu
Popa
Rodica
R
Mashall
Matthew J
MJ
Nguyen
Hien
H
Tebo
Bradley M
BM
Brauer
Suzanna
S
D015373:Bacterial Typing Techniques / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020592:Shewanella / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer