5.7 Terminal Restriction Fragment Length Polymorphism
(T-RFLP)
Terminal restriction fragment duration polymorphism (T-RFLP) overcomes a number of the limitations of RFLP (Thies 2007). T-RFLP is an extension of RFLP
analysis that offers an alternative approach for rapid analysis of microbial network
range in diverse environments. It follows the same precept as RFLP except that one
PCR primer is categorized with a fluorescent dye, together with TET (4,7,2
0 ,70 -tetrachloro-6-carboxyfluorescein) or 6-FAM (phosphoramidite fluorochrome
5-carboxyfluorescein). PCR carried out on sample DNA uses regularly occurring
l6S rDNA primers, one of which is fluorescently categorized. Fluorescently labeled
terminal restriction fragment duration polymorphism (FLT-RFLP) patterns can then
be created by digestion of labeled amplicons using restriction enzymes. Fragments
are then separated by gel electrophoresis on an automated collection analyzer. Each
precise fragment length can be counted as an operational taxonomic unit (OTU), and
the frequency of each OTU may be calculated. The banding sample may be used to
measure species richness and evenness in addition to similarities between samples
(Liu et al. 1997). T-RFLP, as every totally PCR-based approach, may underestimate
authentic range because numerically dominant species are detected by the massive
amount of template DNA (Liu et al. 1997). Incomplete digestion by means of
restriction enzymes can also cause an overestimation of diversity (Osborn et al.
2000). Despite these obstacles, some researchers believe that when standardized,
T-RFLP can be a useful tool to observe microbial variety in the environment (Tiedje
et al. 1999), although others consider it to be inadequate (Dunbar et al. 2000).
T-RFLP has been used to measure spatial and temporal modifications in bacterial
groups (Lukow et al. 2000), to study complex bacterial groups (Moeseneder et al.
1999), to screen populations (Tiedje et al. 1999), and to evaluate the diversity of
arbuscular mycorrhizal fungi (AMF) within the rhizosphere of Viola calaminaria in
a metal-contaminated soil (Tonin et al. 2001). Tiedje et al. (1999) reported five
instances of greater success at detecting and tracking specific ribotypes by using
T-RFLP rather than DGGE.
Dojka et al. (1999) monitored microbial variety in a hydrocarbon- and chlorinated
solvent-infected aquifer undergoing intrinsic bioremediation with T-RFLP, determining sequence types characteristic of Syntrophus spp. and Methanosaeta spp.
They hypothesized from their findings that the terminal step of hydrocarbon degradation inside the methanogenic zone of the aquifer became aceticlastic
methanogenesis, with these organisms existing in a syntrophic relationship.
Bordenave et al. (2004) studied bacterial network modifications in microbial mats
following crude oil pollution using T-RFLP. Their results indicated a clean succession of various bacterial populations with operational taxonomic units that were
associated with the genera Chloroflexus, Burkholderia, Desulfovibrio, and
Cytophaga. T-RFLP has also been used to characterize microbial groups recovered
from surrogate minerals incubated in an acidic uranium-contaminated aquifer
(Reardon et al. 2004) and dechlorinating microorganisms from a basalt aquifer
452
S. K. Shekhar et al.
(T-RFLP)
Terminal restriction fragment duration polymorphism (T-RFLP) overcomes a number of the limitations of RFLP (Thies 2007). T-RFLP is an extension of RFLP
analysis that offers an alternative approach for rapid analysis of microbial network
range in diverse environments. It follows the same precept as RFLP except that one
PCR primer is categorized with a fluorescent dye, together with TET (4,7,2
0 ,70 -tetrachloro-6-carboxyfluorescein) or 6-FAM (phosphoramidite fluorochrome
5-carboxyfluorescein). PCR carried out on sample DNA uses regularly occurring
l6S rDNA primers, one of which is fluorescently categorized. Fluorescently labeled
terminal restriction fragment duration polymorphism (FLT-RFLP) patterns can then
be created by digestion of labeled amplicons using restriction enzymes. Fragments
are then separated by gel electrophoresis on an automated collection analyzer. Each
precise fragment length can be counted as an operational taxonomic unit (OTU), and
the frequency of each OTU may be calculated. The banding sample may be used to
measure species richness and evenness in addition to similarities between samples
(Liu et al. 1997). T-RFLP, as every totally PCR-based approach, may underestimate
authentic range because numerically dominant species are detected by the massive
amount of template DNA (Liu et al. 1997). Incomplete digestion by means of
restriction enzymes can also cause an overestimation of diversity (Osborn et al.
2000). Despite these obstacles, some researchers believe that when standardized,
T-RFLP can be a useful tool to observe microbial variety in the environment (Tiedje
et al. 1999), although others consider it to be inadequate (Dunbar et al. 2000).
T-RFLP has been used to measure spatial and temporal modifications in bacterial
groups (Lukow et al. 2000), to study complex bacterial groups (Moeseneder et al.
1999), to screen populations (Tiedje et al. 1999), and to evaluate the diversity of
arbuscular mycorrhizal fungi (AMF) within the rhizosphere of Viola calaminaria in
a metal-contaminated soil (Tonin et al. 2001). Tiedje et al. (1999) reported five
instances of greater success at detecting and tracking specific ribotypes by using
T-RFLP rather than DGGE.
Dojka et al. (1999) monitored microbial variety in a hydrocarbon- and chlorinated
solvent-infected aquifer undergoing intrinsic bioremediation with T-RFLP, determining sequence types characteristic of Syntrophus spp. and Methanosaeta spp.
They hypothesized from their findings that the terminal step of hydrocarbon degradation inside the methanogenic zone of the aquifer became aceticlastic
methanogenesis, with these organisms existing in a syntrophic relationship.
Bordenave et al. (2004) studied bacterial network modifications in microbial mats
following crude oil pollution using T-RFLP. Their results indicated a clean succession of various bacterial populations with operational taxonomic units that were
associated with the genera Chloroflexus, Burkholderia, Desulfovibrio, and
Cytophaga. T-RFLP has also been used to characterize microbial groups recovered
from surrogate minerals incubated in an acidic uranium-contaminated aquifer
(Reardon et al. 2004) and dechlorinating microorganisms from a basalt aquifer
452
S. K. Shekhar et al.