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L10W10_MIC105_Archaea

L10W10_MIC105_Archaea - similar phylotypes= similar seqs...

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similar “phylotypes”= similar seqs Sunday, January 24, 2010
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Nucleic-acid based hybridizations * Sunday, January 24, 2010
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Sequenced-based approaches to count microbes Indirect counts Most probable number (MPN) Quantitative PCR (QPCR) of specific taxa T-RFLP Direct Counts Light microscopy (phase or staining) Whole cell rRNA-targeted FISH (fluoresecent in situ hybridication) * Sunday, January 24, 2010
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T-RFLP (terminal RFLP) Terminal fragment is fluorescently tagged by PCR Uses one labeled primer Can predict sizes based on database Sunday, January 24, 2010
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Whole cell rRNA-targeted FISH (fluorescence in situ hybridization) • “phylogenetic stain” • ID/count microbes • assess spatial relationships • monitor pop. dynamics (how numbers change/time) • learn cell morphology • “phylotype” vs. phenotype red=FISH, blue=DNA (total) * Sunday, January 24, 2010
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Variable regions tend to make good species-specific probes 5’FITC-gattcgrnat 3’ 3’ ctaagcynta 5’ FITC=fluorescent tag (emits green) * Sunday, January 24, 2010
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A recipe for designing probes Requires a knowledge of phylogenetic relationships and a sequence database (e.g., GenBank) Probes are short 18-35mer oligonucleotides (synthetic DNA), tagged at 5’ terminus with fluorescent group (FITC, Cy3, etc.) Specificity is maximized by adjusting number/placement of mismatches “species”-level generally easier to design than higher phylogenetic levels use rRNA secondary structures * Sunday, January 24, 2010
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red = FISH probe blue = DAPI (DNA) Whole-cell FISH using species-specific probes Sunday, January 24, 2010
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Bacterial divisions TM7: oral cavity sludge Sunday, January 24, 2010
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Sunday, January 24, 2010
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Sunday, January 24, 2010
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Lecture 9: Archaeal Diversity Sunday, January 24, 2010
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Sunday, January 24, 2010
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Carl Woese (U-Illinois) Sunday, January 24, 2010
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MicrobeWorld Podcast “Solving the Puzzle” 4:30 - 11:10 Other relevant podcasts from MicrobeWorld: “The Quest” “Who are we?” “The View from the Forest” Sunday, January 24, 2010
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History of the Archaeal Paradigm Shift Kuhnian paradigm shift Why? Lack of understanding of archaeal biology Genomes and new microbes have helped Ongoing “prokaryote” debate Much research on Archaea in Germany * Sunday, January 24, 2010
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Are Archea “old” or “primitive” Did Archaea “turn into” Eucarya? Are all Archaea “extremophiles?” Are Archaea the “same” as Bacteria Preconceptions about Archaea * Sunday, January 24, 2010
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Archaeal groups have generally been defined phylogenetically Euryarchaeota Crenarchaeota Nanoarchaeota Korarchaeota * Sunday, January 24, 2010
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Dark = includes cultivars Light=uncultivated only Sunday, January 24, 2010
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Alternative, less resolved, view of Archaeal Diversity What’s different?
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