Observation Hypothesis Experimental Design (including Methods) Results Inference Camp Wildness 2004 Ward Lab Research Project

The concept of housekeeping genes ATPase DNA-dependant DNA polymerase DNA-dependant RNA polymerase ribosome components

Ribosome components

Small subunit (16S) rRNA Highly variable segment Highly conserved segment

PCR amplification of SSU rRNA genes Focus on a particular gene in the genome

Extract DNA PCR Mixed 16S rRNA genes Molecular analysis of microbial communities PCR amplification Of 16S rRNA gene Agarose gel analysis of PCR product

Separate by DGGE

Increasing Denaturant Mixed Population DNAs + 16S rRNA Gene G+C “Clamp” PCR PrimersG+C-Tailed Product Separate on Denaturing Gradient Gel PCR Amplification Denaturing Gradient Gel Electrophoresis Separation Based on Differences in Nucleotide Sequence (G+C content) and Melting Characteristics B C Mix A Purified Bands for Sequence Analysis DGGE analysis of 16S rRNA sequences

DGGE study of temperature distribution of Octopus Spring cyanobacterial mat 16S rRNA variants ecotype

Extract DNA PCR Native microbial populations Mixed 16S rRNA Separate by DGGE Sequencing Analyzing distribution of molecular diversity Denaturing Gradient Gel Electrophoresis Phylogenetic identification

Yellowstone cyanobacterial mat cyanobacterial 16S rRNA diversity Readily cultured predominant in situ

A natural view of microbiology

Observation Hypothesis Experimental Design (including Methods) Results Inference Camp Wildness 2004 Ward Lab Research Project