Denaturing gradientgelelectrophoresis (DGGE) is a molecular fingerprinting method that separates polymerase chain reaction (PCR)-generated DNA products. The polymerase chain reaction of environmental DNA can generate templates of differing DNA sequence that represent many of the dominant microbial organisms. However, since PCR products from a given reaction are of similar size (bp), conventional separation by agarose gelelectrophoresis results only in a single DNA band that is largely non-descriptive. DGGE can overcome this limitation by separating PCR products based on sequence differences that results in differential denaturing characteristics of the DNA. During DGGE, PCR products encounter increasingly higher concentrations of chemical denaturant as they migrate through a polyacrylamide gel. Upon reaching a threshold denaturant concentration, the weaker melting domains of the double-stranded PCR product will begin to denature at which time migration slows dramatically. Differing sequences of DNA (from different bacteria) will denature at different denaturant concentrations resulting in a pattern of bands. Each band theoretically representing a different bacterial population present in the community. Once generated, fingerprints can be uploaded into databases in which fingerprint similarity can be assessed to determine microbial structural differences between environments or among treatments (figure below). Furthermore, with the breadth of PCR primers available, DGGE can also be used to investigate broad phylogenies or specific target organisms such as pathogens or xenobiotics degraders.
This study investigated the evolution of archaeal and bacterial populations of two Submerged Anaerobic Membrane Bioreactors (SAMBRs) operating at a mean Solids Residence Time (SRT) of 30 (SAMBR30) and 300 days (SAMBR300) at mesophilic and psychrophilic temperatures. The SAMBRs were fed with leachate produced in a hydrolytic reactor (HR) treating the Organic Fraction of Municipal Solid Waste (OFMSW). The archaeal fingerprint using Denaturing GradientGelElectrophoresis (DGGE) showed different populations in the first and second stage of the two- stage anaerobic process. A build up of Volatile Fatty acids (VFAs) was observed at 20ºC in SAMBR30, while in SAMBR300 the VFAs only built up at 10ºC. The dominant bacterial species in the HR belonged to Prevotella and Thauera, while the dominant ones in SAMBR300 belonged to Sphingobacteriales, Anaerovorax, Spirochaetaceae, Hydrogenophaga, Ralstonia, Prevotella and Smithella. The low bacterial diversity in SAMBR30 compared to SAMBR300 resulted in a persistently high Soluble Chemical Oxygen Demand (SCOD) (>2 g/L) in the bulk reactor due to an insufficient residence time for bacteria to carry out the degradation of recalcitrant COD found in the leachate.
Escherichia coli and other Proteobacteria are augmented and several other bacteria are diminished in Crohn’s (CD) disease patients’ intestine. This imbalance in bacterial species composition—termed dysbiosis—seems to be determinant of CD manifestation. Since a great part of intestinal bacteria are not cultivable, detection of CD dysbiosis is accomplished by molecular tools, involving sequences analysis of the 16SrRNA gene (16SrDNA) present in the patient’s clinical samples, which can be done by sequencing or electrophoresis in denaturing gels of 16SrDNA amplicons. By analyzing, by temperature gradientgelelectrophoresis (TGGE) and next generation sequencing of 16SV6-V8rDNA amplicons present in gram negative cultures from four distinct clinical samples of a control subject and a CD patient, this study demonstrates that both techniques were able to detect E. coli over- growth and reduction in species richness in CD and that TGGE can discriminate sequences collec- tively labeled as “unclassified” in 16SrDNA databases. Although TGGE per se does not identify the sequences, the discriminatory power that it confers represents valuable accessory information to next generation DNA sequencing (NGS), and as such must be used as a NGS complementary tool.
Temporal temperature gradientgelelectrophoresis was first introduced by Yoshino, in 1991. The detection of mutation by TTGE is based on size dependent electrophoretic mobility of double stranded DNA fragmaent in a polyacrylamide gel containing a constant concentration of urea. During electrophoresis, temperature is increased gradually and uniformly. The result is a linear temperature gradient over the electrophoresis run. Thus, a denaturing environment is formed by the constant concentration of urea in the gel in combination with the temporal temperature gradient. In a denaturating environment double stranded DNA is subjected to increase in denaturant environment and will melt in descrete domains, referred to as “Melting Domains”. The melting temperature (Tm) of these domains is sequence specific. When the melting temperature of the lowest domain is reached, the DNA will become partially melted reducing its mobility when compared to wild type DNA. The DNA containing mutations will encounter mobility shifts at different positions in a denaturating gel than the wild type [5-8] .
Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradientgelelectrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n ⴝ 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradientgelelectrophoresis, pyrosequenc- ing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflam- mation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp.
Viridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcus mitis group species (Streptococcus mitis, Strepto- coccus pneumoniae, Streptococcus oralis) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitis group strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitis group strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase (gki) was used in combination with denaturing gradientgelelectrophoresis (DGGE). This gki-PCR-DGGE technique proved to be specific for S. mitis group strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitis group bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitis group strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki-PCR-DGGE technique can track the source of S. mitis group strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemi- ological studies.
Denaturing gradientgelelectrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intra- venous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation be- tween HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate varia- tion in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not cor- related with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.
One approach that is gaining popularity in studies on the diversity of bacteria and archaea natural communities is gradientgelelectrophoresis (GGE), using either a chemical denaturant gradient (DGGE), or a heat gradient (TGGE). This technique, which was originally developed to detect mutations, has been widely applied in studies on prokaryote communities from diverse environments ( Muyzer and Smalla 1998; Galand etal. 2002; Araya etal. 2003; Fasoli etal. 2003; Hein etal. 2003 ). In contrast there has been very few applications of GGE in studies on either freshwater or marine phytoplankton (Van Hannen et al. 1998; Diez etal. 2001) and reliable protocols still need to be developed. In theory, GGE could provide a rapid means of assessing the genetic diversity of microbes in a particular community without the need for detailed taxonomic analysis or establishment of cultures (Dorigo etal. 2002). Here we present results from DGGE methods optimization using clonal laboratory culturesof marine phytoplankton. The DGGE protocols were then applied in a study to compare diversity data obtained for natural marine phytoplankton samples. DGGE variability information obtained was thereafter compared with observations based on traditional microscopy.
PCR-denaturing gradientgelelectrophoresis (DGGE) fingerprinting was first used for profiling complex microbial populations by Muyzer et al. (1993). It has been widely used in environmental microbiology, and is a recognized method for elucidating differences and similarities of dominant populations of microbial communities ( Lindstrom, 2000; Dorigo et al., 2005). In recent years, DGGE has been used to analyze eukaryotic communities using 18S rRNA (Moon-van der Staay et al., 2001; Chen et al., 2011). The present study investigates the nutrient concentrations and eukaryotic plankton diversity in culture ponds with C. auratus gibelio. Canonical correspondence analysis (CCA) methods were used to explore whether there was a close relationship between eukaryotic plankton community succession and
Microtemperature gradientgelelectrophoresis ( -TGGE) was examined for use for the rapid subtyping of Listeria monocytogenes strains. Comparison of genomes between L. monocytogenes strains F2365 and H7858 identified a sequence encoding a portion of the PRT/PTS system IIA 2 protein domain as appropriate for -TGGE analysis. Thirty-one strains belonging to 10 different serovar types were tested by PCR, and sequence analysis of the amplified products revealed that the strains comprise 11 groups. All 55 possible pairs within the 11 groups were examined by -TGGE analysis. Of these, 47 pairs could be successfully discriminated, with a total electrophoresis time of only 7 min. Moreover, Cy3/Cy5 labeling allowed rapid identification of the sequence type in unknown strains of L. monocytogenes isolated from meat. These findings collectively indicate that -TGGE can be used for the rapid analysis of L. monocytogenes strains, facilitating determination of routes of contamination when these bacteria are found in food products.
We used denaturing gradientgelelectrophoresis to detect the beta-thalassemia mutations in the Chinese population. By amplifying the beta-globin gene in four separate fragments and electrophoresing the amplified DNA in two gels, we were able to distinguish all the 12 known mutations on the basis of the mobility of the homoduplexes and heteroduplexes. We conclude that denaturing gradientgelelectrophoresis offers a nonradioactive means of detecting multiple mutations in genetic disorders.
The patchy nature of digestive tract ulceration in CD is unexplained. Postulating that local changes in the microbiota might favor ulceration, we used temporal temperature gradientgelelectrophoresis (TTGE) to compare the qualitative com- positions of the mucosa-associated microbiotas in ulcerated (U) and nonulcerated (NU) regions of the ilea and colons of CD patients. TTGE of 16S rRNA is a powerful technique for comparing the biodiversity of the dominant microbiotas in different biological samples. It is capable of separating bacte- rial sequences with the same size but different thermal stabil- ities (31). Since 16S rRNAs from different bacterial species have different nucleotide sequences in variable regions, their thermal stabilities are also different. This method gives profiles * Corresponding author. Mailing address: Service de Gastro-ente ´r-
Bacteria play an important role in the initiation and progression of periodontal diseases and are part of a biofilm, which can contain over 100 different species. The aim of the present study was to show the potential of denaturing gradientgelelectrophoresis (DGGE) as a tool for the detection of clinically relevant species and to compare the results of detection by DGGE with those by PCR and culturing. Hybridization of the bands from the DGGE profiles with species-specific probes was developed to confirm the band positions in the marker obtained with reference strains. The sensitivities of DGGE compared to those of cultivation for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsyth- ensis were 100, 100, 88, and 100%, respectively; and the sensitivities of DGGE compared to those of PCR were 100, 90, 88, and 96%, respectively. DGGE as a diagnostic tool could easily be extended to other species, as shown for Treponema denticola, which could be detected in 48% of the samples. Three different groups of A. actinomycetemcomitans serotypes could be distinguished by DGGE (i.e., a group comprising serotypes a, d, e, and f; a group comprising serotype b; and a group comprising serotype c). Amplicons from P. gingivalis and T. denticola migrated to the same position in the gel, and P. intermedia produced multiple bands. In the present study we show that the DGGE profiles represent clinically relevant species which can be detected by hybrid- ization with species-specific probes. With DGGE, large numbers of samples can be analyzed for different species simultaneously, and DGGE may be a good alternative in periodontal microbial diagnostics.
Genetic fingerprinting techniques provide a rapid and rela- tively easy alternative to the analysis of microbial communities. The different types of genetic fingerprinting techniques that can be used for this purpose, together with their advantages and disadvantages, have already been described (31). One such fingerprinting technique is the electrophoretic separation of low-molecular-weight rRNA molecules (5S rRNA and tRNA) extracted from natural samples (19). Denaturing gradientgelelectrophoresis (DGGE) is a more recent fingerprinting tech- nique by which PCR-amplified DNA fragments are separated according to their sequence information (33). The basis of this technique is that DNA fragments of the same size but with differing base pair sequences can be separated (32). This sep- aration by DGGE relies on the electrophoretic mobility of partially denatured DNA molecules in a polyacrylamide gel, * Corresponding author. Mailing address: Division of Microbial Dis-
The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradientgelelectrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes Staphylococcus and Pseudomonas spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as Pseudomonas sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.
Previous studies of the endogenous microbiota in patients with ulcerative colitis (UC) have not taken bacterial activity into account, yet bacteria with high transcriptional activity might have a more important pathophysiological role than inactive bacteria. We therefore analyzed the biodiversity of active bacteria in the fecal microbiota of UC patients, in comparison with that of healthy subjects. Feces were collected from nine patients with active UC and from nine healthy controls. Total DNA and RNA were extracted, and 16S ribosomal DNA and RNA were amplified by PCR and reverse transcription-PCR, respectively. Amplification products were compared by means of temporal temperature gradientgelelectrophoresis (TTGE). Bands of interest were excised, sequenced, and identified by comparison with the GenBank database (NCBI). The dominant-species diversity based on RNA-derived TTGE profiles was significantly lower for UC patients than for healthy controls (P ⴝ 0.01). The mean similarity index between the “present” and “active” microbiota was 74% ⴞ 18% for UC patients. Comparison of the individual “active” microbiota identified a band that was present for eight UC patients and only two controls (89% versus 22%; P ⴝ 0.008). The band was sequenced for 6 patients and always corresponded to Escherichia coli. The biodiversity of active bacteria in the dominant fecal microbiota of patients with UC is lower than that of healthy subjects. E. coli is more represented in the active microbiota of UC patients. The possible pathophysiological role of this difference remains to be determined.
To identify some separated and specific bands, a sterile scalpel was used to cut out the bands from polyacryl- amide gel under UV illumination. The gel fragments were washed once in 200 μl of sterile deionized water and kept in 50 μl of sterile water overnight at 4°C for diffusion. The extracted gel mix was heated at 90°C for 10 min, and 4 μl of the solution was taken as the DNA template for re-amplifying by PCR using the original primers without a GC clamp. The PCR program was the same as described previously. After purification, the PCR products were cloned into the PMD18-T Easy vector (TaKaRa, Japan), transformed into competent Escherichia coli Nova blue cells, and screened for posi- tive plasmid insertions according to the manufacturer’s instructions. A second PCR was utilized to confirm the successful construction of the recon. The obtained PCR
Although perhaps the most critical step in the process, the optimization of the initial soil sampling strategy for ﬁngerprinting methods has attracted little attention. Given the possible gel-to-gel variations in DGGE, it is better to limit the quantity of samples to a number that can be analyzed on a single gel or a few gels. This, however, requires a good sample collection strategy, pooling of samples and their size to obtain nucleic acids from representative soil samples. In the soil environment, especially in the top layers of highly stratiﬁed soils, the composition of microbial communities is vertically structured (Kandeler et al. 1999, Baldrian et al. 2008). Changes in microbial community composition occur, even at the centimeter scale within individual soil horizons or among litters of diﬀerent ages (Fioretto et al. 2000, Šnajdr et al. 2008). There is also a consider- able horizontal component of the spatial variability in microbial soil communities at scales of several centimeters to meters (Saetre and Baath 2000, Šnajdr et al. 2008).
In total 5 of the 13 exons of the PAH gene were evaluated for the presence of mutations by DGGE in a group of 50 independent affected patients, living in the South-East of England and attending the University College Hospital London PKU clinic. Each of the exons studied were considered separately. Prior to designing oligonucleotide prim ers for PCR am p lification of genomic DNA, the melting characteristics of each exon with adjacent intron segm ents were evaluated theoretically using the computer algorithms MELT87 and SQHTX, kindly provided by Dr. Leonard Lerman. An artificially high melting domain, in the form of a GC clamp, was introduced at the 5' end of one of the am plification prim ers to allow fo r the id en tificatio n of all potential m utations in the am plified product. The optimum gradient range and electrophoresis time were determined using the SQHTX algorithm for each fragment being investigated.