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Application of Padlock Probe Based Nucleic Acid Analysis In Situ
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Molekylär diagnostik)
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. , p. 41
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 575
Keywords [en]
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: urn:nbn:se:uu:diva-128446ISBN: 978-91-554-7842-1 (print)OAI: oai:DiVA.org:uu-128446DiVA, id: diva2:331818
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: 2018-01-12Bibliographically approved
List of papers
1. Detection of Alu sequences and mtDNA in comets using padlock probes
Open this publication in new window or tab >>Detection of Alu sequences and mtDNA in comets using padlock probes
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2006 (English)In: Mutagenesis, ISSN 0267-8357, E-ISSN 1464-3804, Vol. 21, no 4, p. 243-247Article in journal (Refereed) Published
Abstract [en]

Single cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. However, the behaviour of the DNA under the conditions used for the comet assay is not fully understood. In developing a method for studying specific gene sequences within comets, using 'padlock probes' (circularizable oligonucleotide probes), we have first applied probes that hybridize to Alu repetitive elements and to mitochondrial DNA (mtDNA). During the sequence of stages in the comet assay, mtDNA progressively disperses into the surrounding agarose gel, showing no tendency to remain with nuclear DNA in the comets. In contrast, Alu probes remain associated with both tail and head DNA.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-10568 (URN)10.1093/mutage/gel022 (DOI)000240833700004 ()16940044 (PubMedID)
Available from: 2007-04-04 Created: 2007-04-04 Last updated: 2017-12-11Bibliographically approved
2. Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation
Open this publication in new window or tab >>Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation
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2010 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 195, no 1, p. 31-34Article 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.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-126926 (URN)10.1016/j.toxlet.2010.02.017 (DOI)000278451100005 ()20188804 (PubMedID)
Available from: 2010-06-30 Created: 2010-06-30 Last updated: 2018-01-12Bibliographically approved
3. Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification
Open this publication in new window or tab >>Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification
2011 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 202, no 2, p. 142-147Article 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.

Keywords
Padlock probe, Rolling circle amplification, Comet assay, DNA repair
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-128413 (URN)10.1016/j.toxlet.2011.02.003 (DOI)000289708000009 ()21315812 (PubMedID)
Available from: 2010-07-20 Created: 2010-07-20 Last updated: 2018-01-12Bibliographically approved
4. Different localization of CCHFV vRNA compared cRNA during infection as determined by in situ padlock probe detection
Open this publication in new window or tab >>Different localization of CCHFV vRNA compared cRNA during infection as determined by in situ padlock probe detection
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-128414 (URN)
Available from: 2010-07-20 Created: 2010-07-20 Last updated: 2018-01-12Bibliographically approved

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