Two-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samples

2.50
Hdl Handle:
http://hdl.handle.net/2336/10629
Title:
Two-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samples
Authors:
Gunnarsson, Gudmundur H; Gudmundsson, Bjarki; Thormar, Hans G; Alfredsson, Arni; Jonsson, Jon J
Citation:
Anal. Biochem. 2006, 350(1):120-7
Issue Date:
1-Mar-2006
Abstract:
We describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.
Description:
To access publisher full text version of this article. Please click on the hyperlink in Additional Links field
Additional Links:
http://linkinghub.elsevier.com/retrieve/pii/S0003-2697(05)00887-0

Full metadata record

DC FieldValue Language
dc.contributor.authorGunnarsson, Gudmundur H-
dc.contributor.authorGudmundsson, Bjarki-
dc.contributor.authorThormar, Hans G-
dc.contributor.authorAlfredsson, Arni-
dc.contributor.authorJonsson, Jon J-
dc.date.accessioned2007-03-13T15:21:20Z-
dc.date.available2007-03-13T15:21:20Z-
dc.date.issued2006-03-01-
dc.date.submitted2007-03-13-
dc.identifier.citationAnal. Biochem. 2006, 350(1):120-7en
dc.identifier.issn0003-2697-
dc.identifier.pmid16455036-
dc.identifier.doi10.1016/j.ab.2005.12.013-
dc.identifier.otherGEN12-
dc.identifier.urihttp://hdl.handle.net/2336/10629-
dc.descriptionTo access publisher full text version of this article. Please click on the hyperlink in Additional Links fielden
dc.description.abstractWe describe two-dimensional strandness-dependent electrophoresis (2D-SDE) for quantification and length distribution analysis of single-stranded (ss) DNA fragments, double-stranded (ds) DNA fragments, RNA-DNA hybrids, and nicked DNA fragments in complex samples. In the first dimension nucleic acid molecules are separated based on strandness and length in the presence of 7 M urea. After the first-dimension electrophoresis all nucleic acid fragments are heat denatured in the gel. During the second-dimension electrophoresis all nucleic acid fragments are single-stranded and migrate according to length. 2D-SDE takes about 90 min and requires only basic skills and equipment. We show that 2D-SDE has many applications in analyzing complex nucleic acid samples including (1) estimation of renaturation efficiency and kinetics, (2) monitoring cDNA synthesis, (3) detection of nicked DNA fragments, and (4) estimation of quality and in vitro damage of nucleic acid samples. Results from 2D-SDE should be useful to validate techniques such as complex polymerase chain reaction, subtractive hybridization, cDNA synthesis, cDNA normalization, and microarray analysis. 2D-SDE could also be used, e.g., to characterize biological nucleic acid samples. Information obtained with 2D-SDE cannot be readily obtained with other methods. 2D-SDE can be used for preparative isolation of ssDNA fragments, dsDNA fragments, and RNA-DNA hybrids.en
dc.language.isoenen
dc.publisherAcademic Pressen
dc.relation.urlhttp://linkinghub.elsevier.com/retrieve/pii/S0003-2697(05)00887-0en
dc.subject.meshDNAen
dc.subject.meshDNA, Complementaryen
dc.subject.meshDNA, Single-Strandeden
dc.subject.meshElectrophoresis, Gel, Two-Dimensionalen
dc.subject.meshNucleic Acid Denaturationen
dc.subject.meshNucleic Acid Hybridizationen
dc.subject.meshNucleic Acid Renaturationen
dc.subject.meshRNAen
dc.titleTwo-dimensional strandness-dependent electrophoresis: a method to characterize single-stranded DNA, double-stranded DNA, and RNA-DNA hybrids in complex samplesen
dc.typeArticleen
dc.identifier.journalAnalytical biochemistryen
dc.format.digYES-

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