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Digital quantification of proximity ligation products for highly sensitive and precise protein measurement
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
URN: urn:nbn:se:uu:diva-183138OAI: oai:DiVA.org:uu-183138DiVA: diva2:562017
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2013-01-23
In thesis
1. Detection and Sequencing of Amplified Single Molecules
Open this publication in new window or tab >>Detection and Sequencing of Amplified Single Molecules
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes.

In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them.

Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 829
padlock probes, proximity ligation assay, rolling circle amplification, circle-to-circle amplification, single molecule detection, amplified single molecules, sequencing, in situ, single cell
National Category
Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
urn:nbn:se:uu:diva-183141 (URN)978-91-554-8508-5 (ISBN)
Public defence
2012-12-06, Rudbecksalen, Dag Hammarskjölds v 20, Rudbecklaboratoriet, Uppsala, 09:15 (English)
Available from: 2012-11-15 Created: 2012-10-23 Last updated: 2013-01-23Bibliographically approved

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