with a primer at the 3
0 end of the rrs gene and the other at the
5
0 end of rrl. The approach gives a fingerprint that allows us
to characterize and compare microbial communities
(Fig. 8.2). This technique was applied later (Ranjard
et al. 2000) to the study of soils. A Web of Science search
with RISA as keyword in January 2013 permitted to
retrieve 1,683 references and demonstrated that the use
of RISA remained low during the 2006–2012 period
(Fig. 8.1). This lower use of RISA is probably related to
the fact that the number of intergene sequences available
in databases is much lower (8,788 entries) than that for the
16S (724,000) and 23S (21,427). In actinobacteria, a
hypervariable region at the 5
0 end of the 23S gene can
even be used to make high-resolution phylogeny
(Honerlage et al. 1994) or fine identification of strains
(Roller et al. 1992).
Terminal-Restriction Fragment Length Polymorphism
(T-RFLP)
For this technique DNA is extracted from different
communities to be compared, and a conserved target region
such as 16S rRNA gene is amplified with at least one fluorescent primer. The amplified DNA fragments are then
purified and digested with a restriction enzyme, and the
collection of fragments obtained is then separated by capillary electrophoresis with an automatic sequencer. Each
restriction fragment, or T-RF, corresponds to a sequence
differing from the others by its size, i.e., the distance
between the fluorescent primer and the nearest restriction
site from the fluorescent primer. For instance, this technique
was used to assess the bacterial community dynamics in the
hyporheic zone of an intermittent stream (Febria et al. 2012).
A Web of Science search with T-RFLP as keyword in
December 2012 permitted to retrieve 2,081 references.
An increased use of the method is observed between the
2002–2012 period, though its use remains rather low
(Fig. 8.1).
Fig. 8.1 Temporal variations of
the numbers of papers published
each year which use particular
methods to target genotypic
diversity. The search was done on
the “Web of Science” database
for the 1990–2012 period. All
numbers correspond to absolute
values, except those for
microarrays that were divided per
10. The total number of papers is
7,753, 2,081, 1,683, 10,255,
3,614, 4,728, and 95,838 for
(DGGE and TGGE), T-RFLP,
RISA, SSCP, pyrosequencing,
cloning/sequencing, and
microarrays, respectively
Fig. 8.2 T-RFLP profile of the bacterial community from a microbial
mat. The DNA of all of the microorganisms present is extracted,
purified, and amplified with primers targeting a conserved gene (16S
rRNA), one of which is fluorescent allowing its detection. This composite amplicon is then digested with a restriction enzyme which cuts at
different sites in each amplicon. These gene fragments having a variable length can be separated by electrophoresis, detected by a fluorescence reader, and each peak represents a different sequence whose
amplitude is a function of its abundance in the community
270
P. Normand et al.
0 end of the rrs gene and the other at the
5
0 end of rrl. The approach gives a fingerprint that allows us
to characterize and compare microbial communities
(Fig. 8.2). This technique was applied later (Ranjard
et al. 2000) to the study of soils. A Web of Science search
with RISA as keyword in January 2013 permitted to
retrieve 1,683 references and demonstrated that the use
of RISA remained low during the 2006–2012 period
(Fig. 8.1). This lower use of RISA is probably related to
the fact that the number of intergene sequences available
in databases is much lower (8,788 entries) than that for the
16S (724,000) and 23S (21,427). In actinobacteria, a
hypervariable region at the 5
0 end of the 23S gene can
even be used to make high-resolution phylogeny
(Honerlage et al. 1994) or fine identification of strains
(Roller et al. 1992).
Terminal-Restriction Fragment Length Polymorphism
(T-RFLP)
For this technique DNA is extracted from different
communities to be compared, and a conserved target region
such as 16S rRNA gene is amplified with at least one fluorescent primer. The amplified DNA fragments are then
purified and digested with a restriction enzyme, and the
collection of fragments obtained is then separated by capillary electrophoresis with an automatic sequencer. Each
restriction fragment, or T-RF, corresponds to a sequence
differing from the others by its size, i.e., the distance
between the fluorescent primer and the nearest restriction
site from the fluorescent primer. For instance, this technique
was used to assess the bacterial community dynamics in the
hyporheic zone of an intermittent stream (Febria et al. 2012).
A Web of Science search with T-RFLP as keyword in
December 2012 permitted to retrieve 2,081 references.
An increased use of the method is observed between the
2002–2012 period, though its use remains rather low
(Fig. 8.1).
Fig. 8.1 Temporal variations of
the numbers of papers published
each year which use particular
methods to target genotypic
diversity. The search was done on
the “Web of Science” database
for the 1990–2012 period. All
numbers correspond to absolute
values, except those for
microarrays that were divided per
10. The total number of papers is
7,753, 2,081, 1,683, 10,255,
3,614, 4,728, and 95,838 for
(DGGE and TGGE), T-RFLP,
RISA, SSCP, pyrosequencing,
cloning/sequencing, and
microarrays, respectively
Fig. 8.2 T-RFLP profile of the bacterial community from a microbial
mat. The DNA of all of the microorganisms present is extracted,
purified, and amplified with primers targeting a conserved gene (16S
rRNA), one of which is fluorescent allowing its detection. This composite amplicon is then digested with a restriction enzyme which cuts at
different sites in each amplicon. These gene fragments having a variable length can be separated by electrophoresis, detected by a fluorescence reader, and each peak represents a different sequence whose
amplitude is a function of its abundance in the community
270
P. Normand et al.