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Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2010 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 195, no 1, 31-34 p.Article in journal (Refereed) Published
Abstract [en]

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes.

Place, publisher, year, edition, pages
2010. Vol. 195, no 1, 31-34 p.
National Category
Medical Genetics
URN: urn:nbn:se:uu:diva-126926DOI: 10.1016/j.toxlet.2010.02.017ISI: 000278451100005PubMedID: 20188804OAI: oai:DiVA.org:uu-126926DiVA: diva2:327792
Available from: 2010-06-30 Created: 2010-06-30 Last updated: 2010-12-22Bibliographically approved
In thesis
1. Application of Padlock Probe Based Nucleic Acid Analysis In Situ
Open this publication in new window or tab >>Application of Padlock Probe Based Nucleic Acid Analysis In Situ
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues.

In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair.  The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells.

The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 41 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 575
Padlock probe, rolling circle amplification, single cell gel electrophoresis assay, comet assay, Crimean Congo hemorrhagic fever virus
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
urn:nbn:se:uu:diva-128446 (URN)978-91-554-7842-1 (ISBN)
Public defence
2010-09-10, Fåhreussalen, Rudbeckslaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Available from: 2010-08-20 Created: 2010-07-20 Last updated: 2010-08-31Bibliographically approved

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