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Enhanced molecular analysis by rolling-circle amplification on tag arrays
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
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Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-94682OAI: oai:DiVA.org:uu-94682DiVA: diva2:168628
Available from: 2006-09-01 Created: 2006-09-01 Last updated: 2010-01-13Bibliographically approved
In thesis
1. DNA Tools and Microfluidic Systems for Molecular Analysis
Open this publication in new window or tab >>DNA Tools and Microfluidic Systems for Molecular Analysis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.

Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected.

A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.

A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels.

I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction.

Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 65 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 161
Molecular medicine, Proximity ligation, Microfluidics, Single molecule detection, Microarray, Bonding, Molekylärmedicin
urn:nbn:se:uu:diva-7079 (URN)91-554-6616-8 (ISBN)
Public defence
2006-09-21, Rudbecksalen, C11, Rudbecklaboratoriet, Uppsala, 09:15
Available from: 2006-09-01 Created: 2006-09-01 Last updated: 2013-05-31Bibliographically approved

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