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Microbial Diversity
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Outline 16S/18S rDNA and operational taxonomic units Clone libraries Intergenic Transcribed Spacer Analysis Denaturant Gradient Gel Electrophoresis Terminal Restriction Fragment Length Polymorphism
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Bacterial species are described with chemical (including DNA sequences), physiological, and morphological characteristics
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Bacteria maybe compared through DNA/DNA Hybridization or 16S rDNA sequencing
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DNA/DNA Hybridization
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DNA/DNA Hybridization
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DNA/DNA Hybridization
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DNA sequence analysis The only approach that gives us a detailed description of microbial diversity
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16S ribosomal RNA sequence is often used to determine taxonomic identity of microorganism
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Why use 16S/18S ribosomal gene sequences?
All microorganisms, except viruses have them Slow enough mutation rate that all organisms can be compared
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Comparing DNA sequences
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Comparing DNA sequences
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Phylogenetic trees
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Universal Phylogenetic Tree
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Variation in E.Coli and Archaeal 16S rRNA
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Making clone libraries
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Diversity analysis of deep sea sample
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Generating Fingerprints of Microbial Diversity
TRFLP DGGE RISA
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Maui precipitation gradient
Collaboration with Ted Schuur (University of Florida) Bacterial community profiles Comparison of bacterial community profiles with ecosystem variables
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Hawaiian Precipitation Gradient
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Precipitation Gradient
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Similarities among all 6 sites along the precipitation gradient
Mean Annual Temperature (160C) Dominant Vegetation (Metrosideros forest) Parent Material (Lava and Ash) Substrate Age (410,000 yrs)
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Net Primary Productivity
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N mineralization and Nitrification
g per g day
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Redox Potential (15 cm) Redox Potential (mV)
Mean Annual Precipitation (mm)
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How does the bacterial community change across the precipitation gradient?
Intergenic Transcribed Spacer analysis Denaturant Gradient Gel Electrophoresis Phospho Lipid Fatty Acid Analysis Terminal Restriction Fragment Length Polymorphism analysis
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Terminal Restriction Fragment Length Polymorphism (TRFLP)
PCR 27F R * Digest *
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TRFLP pattern
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TRFLP close-up
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Cluster analysis of TRFLP patterns
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DGGE Principle Low denaturant High Denaturant
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DGGE patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6
Dry Wet Dry Wet
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Cluster Analysis of Denaturant Gradient Gel Electrophoresis patterns
3-2 2-2 5-1 2-1 3-1 1-1 1-2 4-2 4-1 6-1 6-2 5-2
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What is Intergenic Transcribed Spacer Analysis (also known as RISA)?
16S primer 23S primer 16S rDNA Spacer SrDNA
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Intergenic transcribed Spacer Analysis shows bacterial communities between the two soils are different tRNA’s 23SrDNA 16SrDNA Unique spacer sequences
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ITS Patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 M
Dry Wet Dry Wet
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Cluster Analysis of Ribosomal Intergenic Spacer patterns
2-2 1-2 5-1 3-1 4-2 3-2 2-1 4-1 1-1 6-1 6-2 5-2
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effective cation exchange capacity
exchangeable H+ Percent of Maximum Cation Holding Capacity exchangeable Ca2+ exchangeable Al3+ Soil pH
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