Transcript Slide 1

Camp Wildness 2004 Ward Lab Research Project
Observation
Hypothesis
Experimental Design (including Methods)
Results
Inference
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
Molecular analysis
of microbial
communities
PCR amplification
Of 16S rRNA gene
Extract DNA
PCR
Mixed 16S rRNA
genes
Agarose gel analysis
of PCR product
Separate by DGGE
DGGE analysis of 16S rRNA sequences
PCR Amplification
Mixed Population DNAs
16S rRNA Gene
PCR Primers
+
G+C-Tailed Product
G+C “Clamp”
Separate on
Denaturing
Gradient
Gel
Denaturing Gradient Gel Electrophoresis
Purified Bands for
Sequence Analysis
Mix
A
B
C
Increasing
Denaturant
Separation Based on
Differences in
Nucleotide Sequence
(G+C content)
and Melting
Characteristics
DGGE study of temperature distribution of Octopus Spring
cyanobacterial mat 16S rRNA variants
ecotype
Analyzing distribution
of molecular diversity
Denaturing Gradient
Gel Electrophoresis
Native microbial
populations
Extract DNA
PCR
Mixed 16S rRNA
Separate by DGGE
Sequencing
Phylogenetic
identification
Yellowstone cyanobacterial
mat cyanobacterial 16S rRNA
diversity
Pleurocapsa sp.
Phormidium ectocarpi
Synechocystis sp.
Anabaena cylindrica
Spirulina sp.
OS Type I
Oscillatoria amphigranulata
OS C1 Isolate
Readily cultured
Synechococcus sp. 6301
Microcoleus sp.
Oscillatoria sp. 7515
Oscillatoria limnetica
Oscillatoria sp. 6304
OS Type P Isolate
Pseudoanabaena galeata
Gloeobacter violaceus
OS Type J
OS Type B' Isolate
OS Type B Isolate
OS Type A'''
OS Type A''
OS Type A'
OS Type A
OS C9 Isolate
0.01
predominant
in situ
A natural view of microbiology
Camp Wildness 2004 Ward Lab Research Project
Observation
Hypothesis
Experimental Design (including Methods)
Results
Inference