The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

2.50
Hdl Handle:
http://hdl.handle.net/10146/199952
Title:
The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.
Authors:
Ersson, Clara; Moller, Lennart
Abstract:
The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.
Citation:
Mutagenesis 2011, 26 (6):689-695
Journal:
Mutagenesis
Issue Date:
Nov-2011
URI:
http://hdl.handle.net/10146/199952
DOI:
10.1093/mutage/ger034
PubMed ID:
21778357
Additional Links:
http://mutage.oxfordjournals.org/content/26/6/689.long
Type:
Article
Language:
en
ISSN:
1464-3804
Sponsors:
The authors are members of ECVAG which was created within the ECNIS network of excellence in order to validate the comet assay.
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorErsson, Claraen
dc.contributor.authorMoller, Lennarten
dc.date.accessioned2012-01-04T10:38:16Z-
dc.date.available2012-01-04T10:38:16Z-
dc.date.issued2011-11-
dc.identifier.citationMutagenesis 2011, 26 (6):689-695en
dc.identifier.issn1464-3804-
dc.identifier.pmid21778357-
dc.identifier.doi10.1093/mutage/ger034-
dc.identifier.urihttp://hdl.handle.net/10146/199952-
dc.description.abstractThe single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.en
dc.description.sponsorshipThe authors are members of ECVAG which was created within the ECNIS network of excellence in order to validate the comet assay.en
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/content/26/6/689.longen
dc.subjectDNA damageen
dc.subjectDNA migrationen
dc.subjectElectrophoresisen
dc.subjectComet assayen
dc.subjectAgarose densityen
dc.subjectProtocolsen
dc.subject.meshAlkaliesen
dc.subject.meshCell Lineen
dc.subject.meshComet Assayen
dc.subject.meshDNAen
dc.subject.meshDNA Damageen
dc.subject.meshDNA-Formamidopyrimidine Glycosylaseen
dc.subject.meshDose-Response Relationship, Radiationen
dc.subject.meshElectricityen
dc.subject.meshElectrophoresis, Agar Gelen
dc.subject.meshGamma Raysen
dc.subject.meshHumansen
dc.subject.meshHydrogen Peroxideen
dc.subject.meshSepharoseen
dc.titleThe effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.en
dc.typeArticleen
dc.identifier.journalMutagenesisen

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