1. DNA Fingerprinting of Bacterial Communities

2. Overview Targets gene for ribosomal RNA (16S rDNA) Make many DNA copies of the gene for the entire community DNA using modified PCR Cut amplified DNA with restriction enzyme Slight variations in 16S rDNA sequence among the different organisms results in different fragment lengths When analyzed, only the first fragment (length varies for each type of organism) of the 16S rDNA is detected Gives a “fingerprint” of the number of different organisms in a sample (each a different peak) and relative abundance (height of peak) Identity of organism represented by each peak not known

3. T-RFLP Tagged (or Terminal) Restriction Fragment Length Polymorphism fluorescent PCR 16s w/ 1 fluorescent primer Mixed community DNA Digest PCR product with restriction enzyme Labeled fragments of different taxa are different lengths Separate fragments on sequencer Fluorescence detector produces graph of fragments present (Each peak a different type of organism)

4. Example Fingerprint T-RFLP from control site County Brigde

5. Why 16S rDNA? Not all (actually a small percentage) microorganisms can be easily cultured –Culture-based studies are skewed All organisms have ribosomes Function of small subunit RNA (16S in bacteria and archaea) identical in all organisms Regions of varying conservation –Some so conserved they are “universal” –Some so variable they can be used to distinguish between very closely related organisms (different strains of same species)

6. PCR Cool website tutorial Used to amplify a specified region of DNA –Region of DNA specified by “primers” which bind to short sequences of DNA on either end –Primers are short (~18 nucleotide) DNA oligomers

7. Restriction Enzymes Enzymes from bacteria which cut DNA at specific sequences Naturally used by bacteria to protect themselves from foreign DNA (i.e. viruses) Used by biologists like DNA scissors –Useful because you know the sequence where they cut –can differentiate sequences of DNA by different fragment lengths

8. Separating DNA Agarose gel electrophoresis separates DNA fragments by sizeAgarose gel electrophoresis T-RFLP uses capillary electrophoresis –Same principle but 1 nucleotide resolution 12 CATGGTG GTACCACTTAA AATTCGAATTCTTGT GCTTAAGAACA CATGGTG GTACCACTTAA AATTCTTGT GAACA AATTCG GCTTAA

9. Example Fingerprint(s) What do these tell us about the bacterial community? What can’t they tell us? Control (County Bridge)AMD-impacted (DFB099)

10. Dendrogram of Community Similarities More similar T-RFLP patterns on closer branches Control: CB AMD: DFB099 CC FB