PCR-Denaturing Gradient Gel Electrophoresis
The most commonly applied fingerprinting methods used to study the GI-tract microbiota are denaturing and temperature gradient gel electrophoresis (DGGE and TGGE, respectively) of PCR-amplified genes coding for 16S rRNA (Fig. 1) (12,23). Other techniques such as terminal restriction fragment length polymorphism (T-RFLP) and single strand conformation polymorphism (SSCP) analysis are being applied but less frequently (26,29). The common principle of these methods is based on the separation of PCR-amplified segments of 16S rRNA genes of the same length, but with different sequence to visualize the diversity within the PCR amplicons by a banding pattern. One of the PCR primers has a 40-bp GC clamp to hold the DNA strands of the PCR product or amplicon together. With DGGE/TGGE, separation is based on the decreased electrophoretic mobility of partially melted double-stranded DNA molecules in polyacrylamide gels containing a linear gradient of DNA denaturants (a mixture of formamide and urea) or a linear temperature gradient, respectively. As a result mixed amplified PCR products will form a banding pattern after staining that reflects the different melting behaviors of the various sequences (30,31). Subsequent identification of specific bacterial groups or species present in the sample can be achieved either by cloning and sequencing of the excised bands or by hybridization of the profile using phylogenetic probes (30). Furthermore, complementation of the fingerprinting results with statistical analysis provides additional information of the observed diversity by highlighting some putative correlation between different sets of variables (32).
Since its application to study the intestinal microbiota, PCR-DGGE/-TGGE fingerprinting has advanced our knowledge of the intestinal microbiota by unraveling the complexity of this ecosystem and providing insight in the establishment and succession of the bacterial community within the host (23,33). The succession of the microbiota in the feces of infants over the first year of life has been visualized using DGGE profiles of the total microbial community, which showed the relatively simple and unstable infant fecal ecosystem (31). In healthy adults, the predominant fecal microbiota was shown to be complex, host-specific and remarkably stable in time (23,34,35). DGGE profiles for monozygotic twins were significantly more similar than for unrelated individuals, while marital partners showed less similar profiles than twins, indicating the influence of genotype over dietary or environmental factors (35). DGGE profiles also revealed that the predominant bacterial species associated with the colonic mucosa are uniformly distributed along the colon, but significantly different from the predominant fecal community (36,37).
Under certain environmental circumstances and/or in genetically susceptible individuals, there is clear evidence that the GI-tract microbiota may play a role in the pathogenesis and etiology of a number of inflammatory diseases such as ulcerative colitis (UC), and CD (30,38,39). Using DGGE, TGGE and SSCP fingerprinting analyses, it was demonstrated that fecal and mucosal-associated microbiota of patients with UC and CD is altered, less complex, and also unstable over time as compared to matched healthy people (26,40,41). In subjects with irritable bowel syndrome (IBS), higher temporal instability was also seen in comparison to healthy persons, but this was likely influenced by antibiotics used during the study (42).
Bands originating from lactobacilli in fecal samples could not be detected on the DGGE profiles since they represent less than 1% of the community, which is approximately the detection limit of this method (43,44). The dominant fecal microbiota of adults as assessed by DGGE was not significantly altered following consumption of certain probiotic strains (34,43). Although DGGE or TGGE were initially developed for total ecosystem communities, the sensitivity of the method for detecting specific groups that are present in lower numbers in the Gl-tract such as bifidobacteria and especially lactobacilli has been considerably enhanced by using group- or genus-specific primers (34,45-47). Consequently, it was possible to monitor the effect of the administration of prebiotics and/or probiotics on the composition of indigenous bifidobacteria! species, and to track the probiotic strain itself (46). In the latter case, DGGE profiles showed that the simultaneous administration of the prebiotic and probiotic (synbiotic approach) did not improve the colonization of the probiotic strain in the gut of the tested individuals. In another study, the DGGE profiles generated from fecal samples of healthy individuals fed a probiotic strain Lactobacillus paracasei F19, allowed the tracing of the probiotic and supported its presence as autochthonous within the intestinal community of a number of individuals (45). A nested PCR-DGGE approach has been developed to determine the diversity of sulfate-reducing bacteria (SRB) in complex microbial communities (48). SRB have been implicated in the pathogenesis of IBD, and consequently are an interesting population to investigate.
Recently an approach combining GC fractionation with DGGE (GC-DGGE) effectively reduced the complexity of the community DNA mixture being analyzed such that the total diversity within each fraction could be more effectively assessed (49). Thus, initially the total DNA of the complex community was fractionated using buoyant density gradient centrifugation based on the % G + C content, using bisbenzimidazole which preferentially binds to A + T rich regions (50). This fractionation based on G + C content effectively reduced the complexity of the community DNA mixture being analyzed and the total diversity within each fraction could be more effectively assessed by the subsequent DGGE.
Another community fingerprinting technique which is gaining in popularity is T-RFLP (51). The basis is a PCR reaction for the 16S rRNA gene in the complex community followed by restriction enzyme digestion that generates the terminal restriction fragments (T-RFs). The latter are separated by electrophoresis or by using a capillary electrophoresis sequencer, which is more high throughput and reproducible (52), to produce a fingerprint. The technique has been used in several studies, including characterizing the human fecal bifidobacteria, as well as the tracking of probiotic Lactobacillus strains, and monitoring antibiotic-induced alterations in intestinal samples (53,54). Further improvements in this technique include the application of new primer-enzyme combinations for specifically bacterial populations in human feces (29). Furthermore, a novel phylogenetic assignment database for the specific T-RFLP analysis of human fecal microbiota (PAD-HCM) has been designed, which enables a high-level prediction of the terminal-restriction fragments at the species level (55). This will facilitate the use of this technique in studies on the microbiota.
While the application of 16S rDNA-based fingerprinting methods are particularly well suited for examining time series and population dynamics, a more quantitative approach is useful to complement our knowledge about the composition and structure of this complex intestinal ecosystem.
16S rRNA-TARGETED PROBES QUANTIFY THE GI-TRACT MICROBIOTA
Hybridization with rRNA-targeted oligonucleotide probes has become the method of choice for the direct cultivation-independent identification of individual bacterial cells in natural samples. During the last decade, this technique has extended our view of bacterial assemblages and the population dynamics of complex microbial communities (15,56,57). The most commonly used biomarker for hybridization techniques, whether dot-blot or fluorescent in situ hybridization (FISH), is the 16S rRNA molecule because of its genetic stability, domain structure with conserved and variable regions, and high copy number. Highly conserved stretches may thus be used to design domain-specific probes such as EUB338/EUBII /EUBIII which collectively target most of the bacteria, whereas specific probes for each taxonomic level, between bacterial and archaeal, down to genus-specific and species-specific, can be designed according to the highly variable regions of the 16S rRNA (15,58-60). The increasing availability of 16S rRNA sequences has contributed significantly to the development of the hybridization methods and their application in different microbial ecosystems. Unquestionably, the success of the implementation of 16S rRNA hybridization strategies depends on different factors, among them rational design and validation of newly designed rRNA-targeted probes.
There is an online resource for oligonucleotide probes, called probeBase (142), which contains published FISH rRNA-targeted probes as well as recommended conditions of use, and many probes for dominant or interesting microbiota groups are described here (61). When designing new probes, one must consider specificity, sensitivity and accessibility to the target sequence. Nucleic acid probes can be designed to specifically target taxonomic groups at different levels of specificity (from species to domain) by virtue of variable evolutionary conservation of the rRNA molecules. The probes are typically 15-25 nucleotides in length. Appropriate software such as the ARB software package (17) and availability of large databases (http://rdp.cme.msu.edu/html/) are useful tools for rapid probe design and in silico specificity profiling. Additional experimental evaluation of the probes with target and non-target microorganisms is necessary to ensure the specificity and the sensitivity of the newly designed probe. It is important to notice that the validation of a newly designed probe requires different procedures for the dot blot (62) and FISH format (60). Moreover, the hybridization and washing conditions (temperature, salt concentration and detergent) are also crucial for obtaining a detectable probe signal (63). The accessibility of the probe to its target site is another factor to be considered when designing new probes. The accessibility of probe target sites on the 16S and 23S rRNA of Escherichia coli has been mapped systematically by flow cytometry (FCM) and FISH, and it was shown that probe-conferred signal intensities vary greatly among different targets sites (64,65). More recently, it was demonstrated that accessibility patterns of 16S rRNA's are more similar for phylogenetically related organisms; these findings may be the first description of consensus probe accessibility maps for prokaryotes (66).
Nucleic acid probing of complex communities comprises two major techniques: dot blot hybridization and FISH. In the dot blot format, total DNA or RNA is extracted from the sample and is immobilized on a membrane together with a series of RNA from reference strains. Subsequently, the membrane is hybridized with a radioactively labeled probe and
Specific fluorescent oligonucleotides rRNA in ribosomes
Specific fluorescent oligonucleotides
rRNA in ribosomes
Fluorescent microscopy and Image analysis
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