Two-dimensional strandness-dependent electrophoresis

Gunnarsson, Guðmundur H.; Gudmundsson, Bjarki; Thormar, Halldór; Alfredsson, Arni; Jónsson, Jón J.

doi:10.1038/nprot.2006.477

Cited by 8 publications

(8 citation statements)

References 21 publications

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“…In contrast with the results obtained with agarose gels, the electrophoretic mobility of the two strands was indistinguishable in urea–polyacrylamide gels (data not shown); thus the effect described herein has no relevance for the two-dimensional strandedness-dependent electrophoresis approach (17), which is based on PAGE. The possible reasons for the difference between the two gel systems may be related to (i) agarose-specific chemical interactions with ss DNA, (ii) the different average pore size of the two gel types (see above) or (iii) architectural differences in the gel matrix.…”

Section: Resultscontrasting

confidence: 93%

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

Hegedüs

Kókai

Kotlyar

et al. 2009

Nucleic Acids Research

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Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

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Section: Resultscontrasting

confidence: 93%

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

Hegedüs

Kókai

Kotlyar

et al. 2009

Nucleic Acids Research

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“…In order to further confirm the result of this analysis, a twodimensional PAGE experiment [18][19][20] was conducted. It was shown previously that migration of linear and circular nucleic acid species is distinctly dependent on the gel pore size [21].…”

Section: Resultsmentioning

confidence: 87%

RNA self‐processing: Formation of cyclic species and concatemers from a small engineered RNA

Petkovic

Müller

2013

FEBS Letters

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a b s t r a c tWe have engineered a self-processing RNA, derived from the hairpin ribozyme that runs through a cascade of cleavage and ligation reactions thereby changing its topology. The first two cleavage events leave the resulting RNA with a 5 0 -OH group and a 2 0 ,3 0 -cyclic phosphate. Thus, upon refolding, intramolecular ligation delivers a cyclic species. In addition, we demonstrate formation of concatemers resulting from multiple intermolecular ligations. Our results demonstrate the potential of RNA for self-supported topology changes and support the suggestion of 2 0 ,3 0 -cyclic phosphates being suitable activated building blocks for reversible phosphodiester bond formation in the RNA world.

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“…2D-SDE on the minigel platform was done as described by Gunnarsson et al . ( 20 ). Polyacrylamide gels (4%) were made from 30% 29:1 acrylamide:bisacrylamide mixture (Amersham Biosciences) in 1 × tris-borate-EDTA (TBE) and 7 M urea.…”

Section: Methodsmentioning

confidence: 99%

Northern lights assay: a versatile method for comprehensive detection of DNA damage

Gudmundsson

Thormar

Sigurdsson

et al. 2018

Nucleic Acids Research

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DNA damage assays have various limitations in types of lesions detected, sensitivity, specificity and samples that can be analyzed. The Northern Lights Assay (NLA) is based on 2D Strandness-Dependent Electrophoresis (2D-SDE), a technique that separates nucleic acids based on length, strandness, structure and conformation changes induced by damage. NLA is run on a microgel platform in 20–25 min. Each specimen is analyzed in pairs of non-digested DNA to detect single- and double-stranded breaks (DSBs) and Mbo I-digested DNA to detect other lesions. We used NLA to evaluate DNA in solution and isolated from human cells treated with various genotoxic agents. NLA detected and distinguished between single- and DSBs, interstrand and intrastrand DNA crosslinks, and denatured single-stranded DNA. NLA was sufficiently sensitive to detect biologically relevant amount of DNA damage. NLA is a versatile, sensitive and simple method for comprehensive and simultaneous analysis of multiple types of damage, both in purified DNA and in DNA isolated from cells and body fluids. NLA can be used to evaluate DNA quality in biosamples, monitor complex molecular procedures, assess genotoxicity, diagnose genome instability, facilitate cancer theranostics and in basic nucleic acids research.

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Two-dimensional strandness-dependent electrophoresis

Cited by 8 publications

References 21 publications

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

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

RNA self‐processing: Formation of cyclic species and concatemers from a small engineered RNA

Northern lights assay: a versatile method for comprehensive detection of DNA damage

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