Molecular Methods to Measure Intestinal Bacteria: A Review

2012 ◽  
Vol 95 (1) ◽  
pp. 5-23 ◽  
Author(s):  
G Douglas Inglis Agriculture ◽  
Matthew C Thomas ◽  
Dallas K Thomas ◽  
Martin L Kalmokoff ◽  
Stephen P J Brooks ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Intestinal Bacteria ◽  
Polymorphism Analysis ◽  
Sample Collection ◽  
Pcr Bias ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

Abstract The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.

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 Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of soil microbial communities

2009 ◽  
Vol 55 (No. 10) ◽  
pp. 413-423 ◽  
Author(s):  
V. Valášková ◽  
P. Baldrian
Keyword(s):  
Microbial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Microbial Communities ◽  
Soil Microbial ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Experimental Treatments ◽  
Multiple Samples

In soil microbial ecology, the effects of environmental factors and their gradients, temporal changes or the response to specific experimental treatments of microbial communities can only be effectively analyzed using methods that address the structural differences among whole communities. Fingerprinting methods are the most appropriate technique for this task when multiple samples must be analyzed. Among the methods currently used to compare microbial communities based on nucleic acid sequences, the techniques based on differences in the melting properties of double-stranded molecules, denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE), are the most widely used. Their main advantage is that they provide the possibility to further analyze whole sequences contained in fingerprints using molecular methods. In addition to the analysis of microbial communities based on DNA extracted from soils, DGGE/TGGE can also be used for the assessment of the active part of the community based on the analysis of RNA-derived sequences or for the analysis of sequences of functional genes encoding for proteins involved in important soil processes.

Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage

2001 ◽  
Vol 47 (9) ◽  
pp. 829-841 ◽  
Author(s):  
Lisa A May ◽  
Brenda Smiley ◽  
Michael G Schmidt
Keyword(s):  
Fungal Community ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Corn Silage ◽  
Positive Effects ◽  
Operational Taxonomic Units ◽  
Whole Plant ◽  
Lost Productivity ◽  
Gradient Gel Electrophoresis

Significant portions of grain produced for livestock consumption are converted into ensiled forage. Silage producers have long recognized the positive effects of using an inoculant to insure the proper transformation of forage into a palatable and digestible feedstuff. When silage is fed from a storage structure, exposure to air stimulates the growth of epiphytic aerobes that may result in the loss of up to 50% of the dry matter. Moreover, fungi have been found to be associated with ensiled forage, but their growth is normally suppressed by the anaerobic conditions. However, the introduction of oxygen results in a fungal bloom, and the fungi and the associated metabolites may result in lost productivity in the livestock consuming the contaminated forage. In this study, we report on the diversity of the fungal community associated with whole plant corn silage during the ensiling process, and the effect of two different bacterial inoculants as compared with the uninoculated natural epiphytic fermentation on the distribution of the fungi associated with the silage. The fungal community from duplicate mini-silo packages of the same treatment was analyzed by denaturing gradient gel electrophoresis and direct sequencing of the resulting operational taxonomic units. This method proved useful in analyzing the complex microbial communities associated with the forage in that it was possible to determine that one inoculant dramatically influenced the fungal community associated with whole plant corn silage.Key words: fungi, silage, DGGE, OTU.

p53 mutations in ovarian tumors, detected by temperature-gradient gel electrophoresis, direct sequencing and immunohistochemistry

1995 ◽  
Vol 64 (1) ◽  
pp. 52-59 ◽  
Author(s):  
Stefanie Kappes ◽  
Karin Milde-Langosch ◽  
Philipp Kressin ◽  
Babette Passlack ◽  
Barbara Dockhorn-Dworniczak ◽  
...  
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Direct Sequencing ◽  
Ovarian Tumors ◽  
P53 Mutations ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

scholarly journals Detection of mutations in the apolipoprotein CII gene by denaturing gradient gel electrophoresis. Identification of the splice site variant apolipoprotein CII-Hamburg in a patient with severe hypertriglyceridemia

1998 ◽  
Vol 44 (7) ◽  
pp. 1388-1396 ◽  
Author(s):  
Markus S Nauck ◽  
Henrik Nissen ◽  
Michael M Hoffmann ◽  
Jürgen Herwig ◽  
Clive R Pullinger ◽  
...  
Keyword(s):  
Splice Site ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Lipoprotein Metabolism ◽  
Splice Site Mutation ◽  
Site Mutation ◽  
Gradient Gel Electrophoresis ◽  
Detection Of Mutations ◽  
Splice Junctions

Abstract Familial apolipoprotein (apo) CII deficiency is a rare autosomal recessive inborn error of metabolism clinically resembling lipoprotein lipase deficiency. A number of mutations of the apo CII gene are known to date; they are located in the promoter region, the coding exons, or in the splice junctions. We present a simple assay based on PCR and denaturing gradient gel electrophoresis, which allows scanning of the promoter, the entire coding sequence, and the splice junctions of the apo CII gene for sequence variants. All gene fragments are amplified using a common PCR protocol and are examined for mutations on a single gradient gel. Using this method and direct sequencing, we identified homozygosity for a donor splice-site mutation in the second intron, previously designated apo CII-Hamburg, as the genetic cause of apo CII deficiency in a 9-year-old boy presenting with chylomicronemia, eruptive xanthoma, and pancreatitis. In addition, the method allowed us to detect all of six different other known mutations of the apo CII gene. We conclude, therefore, that our assay is highly sensitive; in addition, it is easy to perform and may facilitate the differential diagnosis of disorders of lipoprotein metabolism at the genetic level.

Development of cecal-predominant microbiota in broilers during a complete rearing using denaturing gradient gel electrophoresis

2017 ◽  
Vol 57 (3) ◽  
pp. 458 ◽  
Author(s):  
J. E. Blajman ◽  
M. V. Zbrun ◽  
M. L. Signorini ◽  
J. A. Zimmermann ◽  
E. Rossler ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Probiotic Supplementation ◽  
Immunological Parameters ◽  
Bacterial Dna ◽  
Uncultured Bacteria ◽  
Intervention Measures ◽  
Gradient Gel Electrophoresis ◽  
And Performance

Understanding of the intestinal microbiota is crucial to enhance intestinal health and performance parameters in animals. A more exhaustive research of the intestinal microbiota of broilers could be of interest to implement appropriate intervention measures. The aim of the present study was to investigate the development of the predominant cecal microbiota in broilers that were fed a Lactobacillus salivarius DSPV 001P strain during a complete rearing using denaturing gradient gel electrophoresis (DGGE). Bacterial DNA from cecal samples of 24 broilers at different ages were amplified by PCR and analysed by DGGE. A total of 35 DGGE products were excised and sequenced. Distinctive differences in bacterial communities were observed in the caecum as broilers age. At early stages, identified bacteria within the caecum of broilers were predominantly Clostridium-related species. Also, some sequences had the closest match to the genus Escherichia/Shigella. Furthermore, the caecum was a reservoir rich in uncultured bacteria. The major difference observed in our study was an increase of potentially beneficial Lactobacillus at Day 45. These results may be attributed to modulation of the microbiota by the probiotic supplementation. The obtained data could be relevant for future studies related to the influence of the microbiota resulting from probiotic supplementation on the performance and the immunological parameters of broilers.

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