Transcript DNA Fingerprinting of Bacterial Communities

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