Section 3 update: Denaturing gradient gel electrophoresis (DGGE) in microbial ecology

Muyzer, Gerard; Nübel, Ulrich; Santegoeds, Cecilia M.; Schäfer, Hendrik; Wawer, Cathrin

doi:10.1007/978-1-4020-2177-0_313

Cited by 270 publications

(358 citation statements)

References 73 publications

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“…One-microliter aliquots (*5-10 ng in undiluted form) of each DNA sample was amplified in a 50-ll polymerase chain reaction mix (PCR) comprising 5 ll 109 buffer (Sigma Aldrich Co.), 6 ll of 25 mM MgCl 2 (Sigma Aldrich Co.), 1.2 ll of 20 mg ml -1 BSA (Sigma Aldrich Co.), 0.4 ll of 25 mM dNTP (Sigma Aldrich Co.), 0.5 ll of each primer (20 lM each), 0.2 ll of 5U ll -1 Taq DNA polymerase (Sigma Aldrich Co.), and 35.2 ll of PCRgrade water. Primers were F341 with a GC clamp (5 0 -CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCC TAC GGG AGG CAG CAG-3 0 ) complementary to position 341 to 357 (Escherichia coli numbering), and R907 (CCG TCA ATT CMT TTG AGT TT) complementary to positions 926-907 (Muyzer et al1993(Muyzer et al , 1998. Samples were loaded on a 6 % acrylamide gel with a denaturing gradient of 35-70 % (where 100 % denaturant is 7 M urea and 40 % formamide).…”

Section: Denaturating Gradient Gel Electrophoresis Analysismentioning

confidence: 99%

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

et al. 2015

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Section: Denaturating Gradient Gel Electrophoresis Analysismentioning

confidence: 99%

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

et al. 2015

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“…Conditions for the MMC-specific PCR (primers MMC655f/MMC841r and MMC655f/ 1492r) and details on the development of the PCR conditions are given in the Supplementary Text 1. All DNA samples were prechecked with bacteriaspecific primers 341f and 907r (Muyzer et al, 1998) before specific PCR.…”

Section: Design Of the MMC Pcr Detection Systemmentioning

confidence: 99%

“…To relate the abundance of MMC to total bacteria, a 390-bp fragment of the 16S rRNA gene was amplified with the primer pair 517f and 907r (specific for bacterial 16S rRNA genes, Muyzer et al, 1998) following the protocol described by Sü et al (2006). PCRgenerated and purified 16S rRNA gene fragments of a 3.55-kb plasmid containing the 16S rRNA gene of a MMC phylotype obtained from the German Wadden Sea were applied as standards.…”

Section: Quantitative Pcr Assaysmentioning

confidence: 99%

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

et al. 2011

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Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sedimentassociated bacterial communities.

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“…Denaturing gradient gel electrophoresis (DGGE) and clone library analyses based on 16S rRNA genes were performed using PCR products amplified with primer sets GC341F/907R (Muyzer et al, 1998) and 27F/1492R (Weisburg et al, 1991), respectively. PCR amplifications were also carried out with primers specific for the functional genes mxaF, 1003f and 1555r (Neufeld et al, 2007c); pmoA, A189f and mb661r (Costello and Lidstrom, 1999); mmoX, 206F and 886R (Hutchens et al, 2004); and mauA, mauAf1 and mauAr1 (Neufeld et al, 2007c).…”

Section: Pcr Amplification Of 16s Rrna and Functional Genesmentioning

confidence: 99%

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

et al. 2010

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Lonar Lake is a unique saline and alkaline ecosystem formed by meteor impact in the Deccan basalts in India around 52 000 years ago. To investigate the role of methylotrophy in the cycling of carbon in this unusual environment, stable-isotope probing (SIP) was carried out using the onecarbon compounds methane, methanol and methylamine. Denaturing gradient gel electrophoresis fingerprinting analyses performed with heavy 13 C-labelled DNA retrieved from sediment microcosms confirmed the enrichment and labelling of active methylotrophic communities. Clone libraries were constructed using PCR primers targeting 16S rRNA genes and functional genes. Methylomicrobium, Methylophaga and Bacillus spp. were identified as the predominant active methylotrophs in methane, methanol and methylamine SIP microcosms, respectively. Absence of mauA gene amplification in the methylamine SIP heavy fraction also indicated that methylamine metabolism in Lonar Lake sediments may not be mediated by the methylamine dehydrogenase enzyme pathway. Many gene sequences retrieved in this study were not affiliated with extant methanotrophs or methylotrophs. These sequences may represent hitherto uncharacterized novel methylotrophs or heterotrophic organisms that may have been cross-feeding on methylotrophic metabolites or biomass. This study represents an essential first step towards understanding the relevance of methylotrophy in the soda lake sediments of an unusual impact crater structure.

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Section 3 update: Denaturing gradient gel electrophoresis (DGGE) in microbial ecology

Cited by 270 publications

References 73 publications

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

Microbial community changes along the Ecology Glacier ablation zone (King George Island, Antarctica)

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

Active methylotrophs in the sediments of Lonar Lake, a saline and alkaline ecosystem formed by meteor impact

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