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DNA Fingerprinting of Bacterial Communities 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 T-RFLP Tagged (or Terminal) Restriction Fragment Length Polymorphism PCR 16s w/ 1 fluorescent primer Mixed community DNA Digest PCR product with restriction enzyme Separate fragments on sequencer Fluorescence detector produces graph of fragments present (Each peak a different type of organism) Labeled fragments of different taxa are different lengths Example Fingerprint • T-RFLP from control site County Brigde 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) 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 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 Separating DNA • Agarose gel electrophoresis separates DNA fragments by size • T-RFLP uses capillary electrophoresis – Same principle but 1 nucleotide resolution AATTCGAATTCTTGT GCTTAAGAACA 1 2 CATGGTG GTACCACTTAA AATTCTTGT GAACA CATGGTG GTACCACTTAA AATTCG GCTTAA Example Fingerprint(s) • What do these tell us about the bacterial community? • What can’t they tell us? Control (County Bridge) AMD-impacted (DFB099) Dendrogram of Community Similarities More similar T-RFLP patterns on closer branches Control: CB AMD: DFB099 CC FB