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Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Molekylär diagnostik)
Oslo universitet.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Molekylär diagnostik)
Oslo universitet.
2011 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 202, no 2, 142-147 p.Article in journal (Refereed) Published
Abstract [en]

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

Place, publisher, year, edition, pages
2011. Vol. 202, no 2, 142-147 p.
Keyword [en]
Padlock probe, Rolling circle amplification, Comet assay, DNA repair
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-128413DOI: 10.1016/j.toxlet.2011.02.003ISI: 000289708000009PubMedID: 21315812OAI: oai:DiVA.org:uu-128413DiVA: diva2:330974
Available from: 2010-07-20 Created: 2010-07-20 Last updated: 2017-12-12Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 575
Keyword
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
Identifiers
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)
Opponent
Supervisors
Available from: 2010-08-20 Created: 2010-07-20 Last updated: 2010-08-31Bibliographically approved

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