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.

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.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis

2017 ◽  
Vol 27 (6) ◽  
pp. 350-355 ◽  
Author(s):  
Iva Kotásková ◽  
Barbora Mališová ◽  
Hana Obručová ◽  
Veronika Holá ◽  
Tereza Peroutková ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Urinary Catheter ◽  
Efficient Tool ◽  
Complex Samples ◽  
Operational Taxonomic Units ◽  
Complex Sample ◽  
Gradient Gel Electrophoresis ◽  
Urinary Sample ◽  
Methodological Approaches

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.

High-resolutionHLA-DRBtyping using denaturing gradient gel electrophoresis and direct sequencing

1997 ◽  
Vol 50 (2) ◽  
pp. 170-177 ◽  
Author(s):  
L. A. Knapp ◽  
E. Lehmann ◽  
L. Hennes ◽  
M. E. Eberle ◽  
D. I. Watkins
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Gradient Gel Electrophoresis

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 Double-Gradient Denaturing Gradient Gel Electrophoresis Assay for Identification of L-Ferritin Iron-responsive Element Mutations Responsible for Hereditary Hyperferritinemia-Cataract Syndrome: Identification of the New Mutation C14G

2001 ◽  
Vol 47 (3) ◽  
pp. 491-497 ◽  
Author(s):  
Laura Cremonesi ◽  
Antonella Fumagalli ◽  
Nadia Soriani ◽  
Maurizio Ferrari ◽  
Sonia Levi ◽  
...  
Keyword(s):  
Serum Ferritin ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Large Population ◽  
New Mutation ◽  
Stem Loop ◽  
Iron Responsive Element ◽  
Gradient Gel Electrophoresis ◽  
Responsive Element

Abstract Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5–15% polyacrylamide and 50–95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

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