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Detection of Alu sequences and mtDNA in comets using padlock probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2006 (English)In: Mutagenesis, ISSN 0267-8357, E-ISSN 1464-3804, Vol. 21, no 4, 243-247 p.Article 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.

Place, publisher, year, edition, pages
2006. Vol. 21, no 4, 243-247 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-10568DOI: 10.1093/mutage/gel022ISI: 000240833700004PubMedID: 16940044OAI: oai:DiVA.org:uu-10568DiVA: diva2:38336
Available from: 2007-04-04 Created: 2007-04-04 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Single-molecule Detection in situ
Open this publication in new window or tab >>Single-molecule Detection in situ
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body contains a variety of different cell types that share a common genome, but differ in how they use the information encoded therein. Variation in molecular content exists even at the level of individual cells, and to provide deeper insight into complex cellular processes methods that permit analysis of each cell on its own are needed. This thesis presents molecular methods for localized detection of individual nucleic acid molecules. The developed methods are based on padlock probes and target-primed rolling circle amplification. Single-molecule detection sensitivity in combination with single-nucleotide genotyping selectivity enables detection of allelic DNA variants and closely related target sequences directly in cells. Padlock probes further enable multiplex detection of targets, and in combination with image analysis quantitative molecular data for individual cells can be acquired for large cell populations at a resolution that no other in situ detection method can provide at present.

 

In this thesis, the in situ target-primed rolling circle amplification technique was first used for genotyping of a point mutation in the mitochondrial genome with padlock probes. This displayed mitochondrial DNA heterogeneity in cell populations. Application of the method on comet assay preparations showed that mitochondrial genomes are lost from these samples prior to analysis. Nuclear DNA targets, however, can be efficiently detected in corresponding samples. Padlock probes and rolling circle amplification are thus an attractive alternative to FISH analysis for localized DNA detection in comet assay samples. A method was also developed for localized detection of individual mRNA molecules with padlock probes and rolling circle amplification. This method provides unique possibilities to genotype allelic variants of transcripts in situ. mRNA expression is associated with substantial cell-to-cell variation and our presented method permits simultaneous visualization of multiple transcripts directly in complex tissue samples. Application of the methods presented in this thesis will enable new types of studies of biological samples from both normal and disease states.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 431
National Category
Cell and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-98538 (URN)978-91-554-7442-3 (ISBN)
Public defence
2009-04-09, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-03-19 Created: 2009-02-25 Last updated: 2009-05-18Bibliographically approved
2. 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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Larsson, ChatarinaHenriksson, SaraNilsson, Mats

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