Bacterial community analysis using temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA PCR products of soil metagenome

Ekologija ◽  
2010 ◽  
Vol 56 (3) ◽  
pp. 94-98 ◽  
Author(s):  
Ravindra Soni ◽  
Bhoomika Saluja ◽  
Reeta Goel
Keyword(s):  
Temperature Gradient ◽  
Bacterial Community ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Community Analysis ◽  
16S Rdna Pcr ◽  
Soil Metagenome ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

scholarly journals Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

2007 ◽  
Vol 74 (1) ◽  
pp. 188-199 ◽  
Author(s):  
Sara Beier ◽  
Karl-Paul Witzel ◽  
Jürgen Marxsen
Keyword(s):  
Sequence Analysis ◽  
Temperature Gradient ◽  
Bacterial Community ◽  
Community Composition ◽  
Gel Electrophoresis ◽  
Stream Water ◽  
Bacterial Community Composition ◽  
Water And Sediment ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

ABSTRACT The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.

scholarly journals Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis

1999 ◽  
Vol 45 (8) ◽  
pp. 1162-1167 ◽  
Author(s):  
Tian-Jian Chen ◽  
Richard G Boles ◽  
Lee-Jun C Wong
Keyword(s):  
Mitochondrial Dna ◽  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Direct Sequencing ◽  
Polymorphism Analysis ◽  
Mtdna Mutations ◽  
Sequence Variations ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

Abstract Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations. Methods: DNA samples were selected from patients with known mtDNA mutations and patients suspected of mtDNA disorders without detectable mutations by routine analysis. Six regions of mtDNA were PCR amplified and analyzed by TTGE. Electrophoresis was carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR/allele-specific oligonucleotide or PCR/restriction fragment length polymorphism analysis. Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic polymorphism. In 109 specimens with 15 different known mutations, TTGE detected the presence of all mutations and distinguished heteroplasmic mutations from homoplasmic polymorphisms. When 11% of the mtDNA genome was analyzed by TTGE in 104 patients with clinically suspected mitochondrial disorders, 7 cases of heteroplasmy (≈7%) were detected. Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to be a sensitive tool for detection of sequence variations and heteroplasmy in patients suspected of having mtDNA disorders.

scholarly journals Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria

1998 ◽  
Vol 64 (10) ◽  
pp. 3854-3859 ◽  
Author(s):  
Erwin G. Zoetendal ◽  
Antoon D. L. Akkermans ◽  
Willem M. De Vos
Keyword(s):  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Human Gastrointestinal Tract ◽  
Banding Patterns ◽  
Fecal Samples ◽  
Gradient Gel Electrophoresis ◽  
Preferential Amplification ◽  
Temperature Gradient Gel Electrophoresis

ABSTRACT The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from differentClostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.

Root nodules ofCeanothus caeruleuscontain both the N2-fixingFrankiaendophyte and a phylogetically related Nod-/Fix-actinomycete

1998 ◽  
Vol 44 (2) ◽  
pp. 140-148 ◽  
Author(s):  
Hugo Ramírez-Saad ◽  
Jaap D Janse ◽  
Antoon DL Akkermans
Keyword(s):  
Temperature Gradient ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Root Nodules ◽  
16S Rdna Sequence Analysis ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
The Family ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

Attempts to isolate the N2-fixing endophyte of Ceanothus caeruleus (Rhamnaceae) root nodules, led to the isolation of nine actinomycetous strains. Owing to their inability to fix nitrogen (Fix-) and nodulate (Nod-), they could not be regarded as the effective endophyte. Characterization was done based on morphological and physiological features and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and sequencing of nearly full length 16S rDNA and partial nifH genes. Phylogenetic analysis based on 16S rDNA revealed that both the effective endophyte and the isolated actinomycetes belong to two different but well-defined lineages within the family Frankiaceae. One lineage is formed mainly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix-/Nod-isolates. Application of temperature gradient gel electrophoresis techniques to actinorhizal nodules allowed us to detect and identify 16S rDNA sequences from both the Fix+and the Fix-nodule inhabitants. Interestingly, these same two sequences were detected on Hippophae rhamnoides nodules obtained after inoculation with Ceanothus caeruleus nodule suspensions. The isolates were located in the outer layers of the nodule.Key words: Frankia, Ceanothus, 16S rDNA, nifH, temperature gradient gel electrophoresis (TGGE), Fix-/Nod-strains.

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