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Detection and Sequencing of Amplified Single Molecules
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Mats Nilsson)
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 829
Keyword [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-183141ISBN: 978-91-554-8508-5 (print)OAI: oai:DiVA.org:uu-183141DiVA: diva2:562020
Public defence
2012-12-06, Rudbecksalen, Dag Hammarskjölds v 20, Rudbecklaboratoriet, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-11-15 Created: 2012-10-23 Last updated: 2013-01-23Bibliographically approved
List of papers
1. Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
Open this publication in new window or tab >>Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
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2011 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 49, no 12, 4279-4285 p.Article in journal (Refereed) Published
Abstract [en]

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-167925 (URN)10.1128/JCM.00713-11 (DOI)000298113400041 ()
Available from: 2012-02-02 Created: 2012-02-02 Last updated: 2017-12-08Bibliographically approved
2. Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
Open this publication in new window or tab >>Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2, e31068- p.Article in journal (Refereed) Published
Abstract [en]

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-174597 (URN)10.1371/journal.pone.0031068 (DOI)000302875500012 ()
Available from: 2012-05-24 Created: 2012-05-22 Last updated: 2017-12-07Bibliographically approved
3. Digital quantification of proximity ligation products for highly sensitive and precise protein measurement
Open this publication in new window or tab >>Digital quantification of proximity ligation products for highly sensitive and precise protein measurement
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:uu:diva-183138 (URN)
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2013-01-23
4. Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
Open this publication in new window or tab >>Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads
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2012 (English)In: Small, ISSN 1613-6810, Vol. 8, no 14, 2174-2177 p.Article in journal (Refereed) Published
Abstract [en]

A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathogenic simulant of Bacillus anthracis, with a limit-of-detection of 50 spores with a reaction time of 135 min. The study shows the versatility of magnetic nanobeads for detection of biological molecules other than DNA.

Keyword
biosensors, immunoassays, magnetic particles, spores, nanobeads
National Category
Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials; Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-169224 (URN)10.1002/smll.201102632 (DOI)000306362700006 ()22514097 (PubMedID)
Available from: 2012-02-24 Created: 2012-02-24 Last updated: 2016-11-30Bibliographically approved
5. In situ sequencing of mRNA in intact cells and tissues
Open this publication in new window or tab >>In situ sequencing of mRNA in intact cells and tissues
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-183140 (URN)
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2013-01-23

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