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Profiling microbial communities
with T-RFLP
(terminal restriction fragment length
polymorphism)
Anne Fahy
why microbial ecology?
Microbial organisms occupy a peculiar place in the human view of life.
Microbes receive little attention in our general texts of biology. They are
largely ignored by most professional biologists and are virtually unknown
to the public except in the context of disease and rot. Yet, the workings of
the biosphere depend absolutely on the activities of the microbial world.
(Pace, 1997)
 huge metabolic diversity
 “higher” organisms dependent on microbial activities
 applications: bioremediation and natural attenuation
of pollutants in the environment
why culture-independent?
 between 0.001 and 1 %
microorganisms are culturable
(Amann 1995)
 microbial communities are
complex:
huge diversity
close interactions between
organisms
highly dynamic
phylogenetic tree based on 16S rRNA : major phyla
of the domain Bacteria (Rappé & Giovannoni, 2003)
black = 12 original phyla described by Woese, 1987
white = 14 phyla with isolated representatives
grey = 26 candidate phyla with no known isolates
why 16S rRNA as a
phylogenetic marker ?
- protein translation : universal
- no horizontal transfer
(caveat: Wang & Zhang, 2000)
- convenient length : 1500 bp
- highly conserved regions as
well as species-specific regions
- large databases (EMBL, NCBI,
DDJB)
secondary structure of the Escherichia coli
16S rRNA molecule (Van de Peer, et al. 1996).
colours  variability between organisms:
pink = highly conserved
red = least conserved
grey = unaligned
other phylogenetic markers
- proteins; difficult to identify homologous proteins
(Demoulin, 1979)
- historically: also 5S, 23S rRNA
- ribosomal intergenic spacer: 16S – 23s
- 18S for Eukaryotes
T-RFLP
(Terminal Restriction Fragment Length Polymorphism)
1 extraction of community DNA or RNA from environmental sample
(need RT-PCR step with RNA)
3 digestion of amplicons
with restriction enzyme
2 PCR amplification of 16S
rRNA gene with fluorescent
primers
4 detection and sizing of labelled
terminal fragments by capillary
or gel electrophoresis
raw data
Red: internal size standard
T-RFLP (2)
Blue: forward primer
Green: reverse primer
T-RFLP (3)
Analysis of raw data with Genescan:
virtual filter: adjust overlap of fluorescence
sizing: standard curve
integration of peaks
size calling curve
T-RFLP (4)
T-RF length in nucleotides
relative
peak
height
Electropherogram: a visual
profile of the community.
In principle, the height and area of
the peaks are representative of the
abundance of the groups of
organisms.
Several groups of organisms may
share the same T-RF.
Table:
digital data can be further
processed and used, for example, to
generate dendrograms illustrating the
relationship
between
bacterial
communities.
T-RFLP : resolution
- several groups of organisms may share the same T-RF
- T-RFs need to be within range of size standard
resolution of T-RFLP depends on the choice
of restriction enzyme / primer combination
Ribosome Database Project:
 TAP-TRFLP application
 enter choice of enzyme/primer
 in silico digestion of 16S rRNA on the database
http://rdp8.cme.msu.edu/html/TAP-trflp.html#program
several digests + combine data  increase resolution
T-RFLP : good technique
- reproducible technique
- relatively fast  monitor community dynamics
- culture-independent
- digital data for further analyses
- link data to clone libraries
Rs + PO4
Rhodoferax antarcticus ( Proteobacteria, Comamonadaceae)
but…..
…. need to look at data to avoid pitfalls and know the limitations
sources of biases (von Wintzingerode et al., 1997):
- experimental design
- sampling
- storage of sample
- DNA extraction
- PCR amplification (loads of literature!)
keep experimental procedures constant
 PCR-based techniques provide information that is not
obtainable through other methods
limitations inherent to T-RFLP
- glitches in the electrophoresis  rerun sample
- incomplete digestion (partially single-stranded amplicons; Egert
& Friedrich, 2003)  be aware
clone M232: 3 hours digestion
clone M232: 15 hours digestion
limitations inherent to T-RFLP (2)
renaturation of sample:
salts in buffer
amount of DNA in sample
delay between denaturation and electrophoresis
renaturation of internal size standard
 rerun sample
limitations inherent to T-RFLP (3)
overloading of the capillary  rerun sample
12 seconds injection
d
d
6 seconds injection
3 seconds injection
limitations inherent to T-RFLP (4)
sizing problems
discrepancy between:
expected T-RF (from in silico digestion of known sequence)
apparent T-RF (from electrophoresis)
 caution when interpreting T-RFLP profiles
limitations inherent to T-RFLP (5)
sizing problems
causes:
- apparent size varies with the type of genetic analyser: a 142 nt
fragment will measure 143.4 and 140.6 nt respectively on a gel or
capillary genetic analyser (GeneScan Reference Guide)
- resolution: decreases as fragment length increases
- ROX label of internal standard migrates more slowly than the FAM
label of the forward primer (Boorman et al., 2002)
- apparent size of fragment depends on its secondary structure
limitations inherent to T-RFLP (6)
sizing problems
12
Difference in size (nt)
10
8
6
4
2
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
Expected fragm ent size in nucleotide (CfoI )
- difference ± proportional to fragment length
- can vary from -2 to -4 nt for very similar length of fragment
- outside range of size standard: can’t size accurately
- abnormal migration
375
400
425
limitations inherent to T-RFLP (7)
sizing problems
very abnormal migration from a specific clone T-RF:
expected size
apparent size
difference
AluI
109
105.8
3.2
CfoI
114
103.0
11.0
- possible “hairpin” from secondary structure
-----CGGAACGTGCCCAGTCGTGGGGGATAACGCAGC G
------CGGAACGTGCCCAGTCGTGGGGGATAACGCAGCGA
------CGGAACGTGCCCAGTCGTGGGGGATAA GCGTCGGA
------CGGAACGTGCCCAGTCGTGGGGGATAAGCGTCGA
- no such discrepancy from other clones with same sequence
immediately preceding the restriction site
in press: Nogales et al.
(a study of mobility anomalies of 16S rRNA gene fragments)
limitations inherent to T-RFLP (8)
Conclusions:
T-RFLP very reproducible (electropherograms need to be perfect)
comparison of data limited to studies using same type of genetic
analyser
cannot predict phylogenetic affiliations from the length of the T-RFs
within its limitations, T-RFLP is a good culture-independent
technique for profiling microbial communities!
many community profiling techniques
Techniques based on PCR of rDNA
Cloning and sequencing of 16S rDNA
DGGE (denaturing gradient gel electrophoresis)
SSCP (single strand conformation polymorphism)
RFLP (restriction fragment length polymorphism)
LH-PCR (length heterogeneity analysis by PCR)
ARISA (automated ribosomal intergenic spacer analysis)
DGGE and T-RFLP also used for diversity of catabolic genes
Other approaches to community profiling
Hybridisation, FISH, PLFA, BIOLOG
Linking metabolic function to phylogeny
SIP (stable isotope probing)