1. Analysis of Microbial Community Structure
Historical Perspective on Microbial Diversity
Assessing Diversity Alone
Diversity With Phylogeny
Diversity of Function

2. Historical Perspective on Microbial Diversity
Knowledge is limited by our tools.
Cultivation missed about 99.9% of no. estimates.
Both biomass and diversity underestimated.
Molecular techniques and phylogeny:
Independent of cultivation
Unknowns can be grouped with knowns

3. Study Diversity Independent of Cultivation
Extract DNA from sample (or not?)
PCR Amplification of 16SrDNA
Clone all amplicons
Screen clones for differences (e.g. ARDRA).
Document richness and evenness of clones.
Preliminary phylogeny
Sequence clones of interest.
Perform complete phylogenetic analysis; & identify known or prior documented unknown strains.

5. Ligate PCR amplicon into a cloning plasmid; transform host bacterium; isolate recombinant plasmids for sequencing of inserted 16SrDNA.

6. Screen Clones ARDRA is an RFLP on the 16SrDNA amplified by PCR
RFLP = restriction fragment length polymorphism
ARDRA has been used:
- post-cloning
pre-cloning
isolates or communities
For screening clones with 16SrDNA, enzymes that separate plasmid DNA are used for clearer results.

7. Preliminary Phylogenetic Analysis
Fingerprints (banding patterns) can be converted to analog data based on presence or absence of bands of different sizes.
Pairwise comparison of all clone fingerprints will yield a similarity (distance) matrix.
Phylogenic tree can be computed from a cluster analysis of the matrix.

8. Complete Analysis 16SDNA Sequencing:
1) Dideoxynucleotides stop synthesis at different sites; different size fragments made for each sequence position.
2) Each ddNTP has a fluorescent label for easy specific detection as it’s separated by HPLC or electrophoresis.

9. Quick Assessment of Diversity “One band = One bug” (
Quick Assessment of Diversity “One band = One bug” (?) (little phylogeny information)
RISA (ribosomal intergenic spacer analysis)
Often get overlapping bands (on band = > 1 bug)
Phylogenetic information limited by 16SrDNA overlap
ARDRA (amplified ribosomal DNA restriction analysis)
Good for identification of isolates; esp. with multiple restriction enzymes.
Too many bands makes it hard to interpret mixed populations.
T-RFLP (terminal restriction fragment polymorphism)
Steps like ARDRA, but terminal 3’ end of gene is fluorescent
Multiple restriction enz. Give best results; maybe used to query RDP.

10. DGGE Denaturing Gradient Gel Electrophoresis
Separated DNA of same size based on sequence differences.
Different sequences “behave differently at different amounts of denaturing chemical (or heat; see TGGE)
At some point 16SrRNA DNA strands completely separates.
Complete separation of PCR amplicon is hindered by GC-clamp added to one of the PCR primers.

11. Gradient Perpendicular to Electrophoresis to Optimize Run

12. Gradient Parallel for Analysis
Conditions change mostly due to size of amplicon.
May be applied at different taxa (groups)
Bands may be cut out and DNA cloned for phylogenetic analysis.
Larger bands more information.

13. Diversity Metrics:
Shannon-Weaver
Simpson