Microbio 310 1st Edition Lecture 20 Outline of Last Lecture I. 16.1 Formation and Early History of Earth II. 16.2 Origin of Cellular Life III. 16.3 Microbial Diversification: Consequences for Earth’s Biosphere IV. 16.4 Endosymbiotic Origin of Eukaryotes V. 16.5 The Evolutionary Process VI. 16.6 Evolutionary Analysis: Theoretical Aspects VII. 16.7 Evolutionary Analysis: Analytical Methods Outline of Current Lecture I. 16.9 Applications of SSU rRNA Phylogenetic MethodsII. 16.10 Phenotypic AnalysisIII. 16.11 Genotypic AnalysisCurrent Lecture16.9 Applications of SSU rRNA Phylogenetic Methods• Signature sequences– Short oligonucleotides unique to certain groups of organisms– Often used to design specific nucleic acid probes • Probes– Can be general or specific– Can be labeled with fluorescent tags and hybridized to rRNA in ribosomes within cellsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.• FISH: fluorescent in situ hybridization– Circumvent need to cultivate organism(s)• PCR can be used to amplify SSU rRNA genes from members of a microbial community– Genes can be sorted out, sequenced, and analyzed– Such approaches have revealed key features of microbial community structure and microbial interactions• Ribotyping – Method of identifying microbes from analysis of DNA fragments generated from restriction enzyme digestion of genes encoding SSU rRNA; then use electrophoresis– Highly specific and rapid– Used in bacterial identification in clinical diagnostics and microbial analyses of food, water, and beverage (Do yogurts really contain the microbes the package says they have?)16.10 Phenotypic Analysis• Bacterial taxonomy incorporates multiple methods for identification and description of new species• The polyphasic approach to taxonomy uses three methods:1. Phenotypic analysis Fatty acid typing (what it looks like, where it lives)2. Genotypic analysis 3. Phylogenetic analysis (compare sequence data to other species/genus)16.11 Genotypic Analysis• Several methods of genotypic analysis are available:– DNA–DNA hybridization– DNA profiling– Multilocus sequence typing (MLST)– GC ratio• DNA–DNA hybridization (mix DNA from different organisms and see if they hybridize)– Provides rough index of similarity between two organisms– Useful complement to SSU rRNA gene sequencing– Useful for differentiating very similar organisms– Hybridization values of 70% or higher suggest strains belong to the same species• Values of at least 25% suggest same genus• DNA profiling– Several methods can be used to generate DNA fragment patterns for analysis of genotypic similarity among strains (DNA fingerprinting), including:• Ribotyping: focuses on a single gene- Repetitive extragenic palindromic PCR (rep-PCR) and amplified fragment length polymorphism (AFLP): focus on many genes located randomly throughout genome• Multilocus sequence typing (MLST)– Method in which several different “housekeeping genes” from an organism are sequenced– Has sufficient resolving power to distinguish between very closely related strains• GC ratios– Percentage of guanine plus cytosine in an organism’s genomic DNA– Vary from 20 to 80% among Bacteria and Archaea– Generally accepted that if GC ratios of two strains differ by ~5% they are unlikely to be closely
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