Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Hegedüs, Éva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

doi:10.1093/nar/gkp539

Cited by 19 publications

(27 citation statements)

References 29 publications

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“…Next we prepared our own λ‐phages (supplementary note S2.1 & figure S5) and compared the quality of the genomic DNA to the commercially available λ‐DNA. Denaturing agarose gels showed that our ‘homemade’ λ‐DNA directly released from phage particles is intact (supplementary note S2.2 & figure S6) …”

Section: Methodsmentioning

confidence: 99%

DNA Origami Structures Directly Assembled from Intact Bacteriophages

Nickels

Jungmann

et al. 2014

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Section: Methodsmentioning

confidence: 99%

DNA Origami Structures Directly Assembled from Intact Bacteriophages

Nickels

Jungmann

et al. 2014

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“…In this updated protocol, we use agarose-urea gels to perform denaturing gel electrophoresis [ 23 ]. A detailed protocol using alkaline gel electrophoresis can be found in the fi rst edition of this chapter [ 20 ].…”

Section: Notesmentioning

confidence: 99%

Methods to Study Transcription-Coupled Repair in Chromatin

Gaillard¹,

Wellinger²,

Aguilera³

2015

Methods in Molecular Biology

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The effect of endogenous and exogenous DNA damage on the cellular metabolism can be studied at the genetic and molecular level. A paradigmatic case is the repair of UV-induced pyrimidine dimers (PDs) by nucleotide excision repair (NER) in Saccharomyces cerevisiae. To follow the formation and repair of PDs at specific chromosome loci, cells are irradiated with UV-light and incubated in the dark to allow repair by NER. Upon DNA isolation, cyclobutane pyrimidine dimers, which account for about 90 % of PDs, can be cleaved in vitro by the DNA nicking activity of the T4 endonuclease V repair enzyme. Subsequently, strand-specific repair in a suitable restriction fragment is determined by denaturing gel electrophoresis followed by Southern blot and indirect end-labeling using a single-stranded DNA probe. Noteworthy, this protocol could potentially be adapted to other kind of DNA lesions, as long as a DNA nick is formed or a lesion-specific endonuclease is available.Transcription-coupled repair (TC-NER) is a sub-pathway of NER that catalyzes the repair of the transcribed strand of active genes. RNA polymerase II is essential for TC-NER, and its occupancy on a damaged template can be analyzed by chromatin immunoprecipitation (ChIP). In this chapter, we provide an up-dated protocol for both the DNA repair analysis and ChIP approaches to study TC-NER in yeast chromatin.

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“…Urea-agarose gel electrophoresis The procedure to prepare and run the samples in urea-agarose gel electrophoresis was adapted from Hegedüs et al (2009) with minor modifications. Before loaded on the gels, samples were urea-/heat-denatured.…”

Section: Screening Of Cloned Amplicons By Urea-agarose Gel Electrophomentioning

confidence: 99%

“…Aiming to avoid sequencing all cloned products selected for each consortium, the simple and sensitive DNA fingerprinting analysis method based on urea-agarose gel electrophoresis described by Hegedüs et al (2009) was tested and applied to identify similar cloned amplicons.…”

Section: Screening Of Cloned Amplicons By Urea-agarose Gel Electrophomentioning

confidence: 99%

“…Then, having only three samples, and because the aim was to identify the main groups of bacteria that constitute the bulk of the communities and not rare taxa, we characterized the bacteria consortia based on 16S rRNA gene sequences selected following the above considered classical approach. To that purpose, a new simple and sensitive method to detect polymorphisms based on urea-agarose gel electrophoresis (Hegedüs et al 2009) was tested and applied to select representative cloned amplicons for Sanger sequencing in order to taxonomically classify their origin and thus characterize the major bacterial groups in the consortia. Moreover, we compared the taxonomic classifications retrieved with sequences from one half side of the 16S rRNA gene with classifications obtained with sequences from the other half side of the gene obtained by sequencing cloned amplicons with two universal plasmid primers flanking the cloning site, to study the feasibility of using only one of the primers to reduce sequencing costs.…”

Section: Introductionmentioning

confidence: 99%

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Application of urea–agarose gel electrophoresis to select non-redundant 16S rRNAs for taxonomic studies: palladium(II) removal bacteria

Assunção

Costa

Carlier

2015

Appl Microbiol Biotechnol

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The 16S ribosomal RNA (rRNA) gene has been the most commonly used sequence to characterize bacterial communities. The classical approach to obtain gene sequences to study bacterial diversity implies cloning amplicons, selecting clones, and Sanger sequencing cloned fragments. A more recent approach is direct sequencing of millions of genes using massive parallel technologies, allowing a large-scale biodiversity analysis of many samples simultaneously. However, currently, this technique is still expensive when applied to few samples; therefore, the classical approach is still used. Recently, we found a community able to remove 50 mg/L Pd(II). In this work, aiming to identify the bacteria potentially involved in Pd(II) removal, the separation of urea/heat-denatured DNA fragments by urea-agarose gel electrophoresis was applied for the first time to select 16S rRNA-cloned amplicons for taxonomic studies. The major raise in the percentage of bacteria belonging to genus Clostridium sensu stricto from undetected to 21 and 41 %, respectively, for cultures without, with 5 and 50 mg/L Pd(II) accompanying Pd(II) removal point to this taxa as a potential key agent for the bio-recovery of this metal. Despite sulfate-reducing bacteria were not detected, the hypothesis of Pd(II) removal by activity of these bacteria cannot be ruled out because a slight decrease of sulfate concentration of the medium was verified and the formation of PbS precipitates seems to occur. This work also contributes with knowledge about suitable partial 16S rRNA gene regions for taxonomic studies and shows that unidirectional sequencing is enough when Sanger sequencing cloned 16S rRNA genes for taxonomic studies to genus level.

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Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Cited by 19 publications

References 29 publications

DNA Origami Structures Directly Assembled from Intact Bacteriophages

DNA Origami Structures Directly Assembled from Intact Bacteriophages

Methods to Study Transcription-Coupled Repair in Chromatin

Application of urea–agarose gel electrophoresis to select non-redundant 16S rRNAs for taxonomic studies: palladium(II) removal bacteria

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