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A dual-tag microarray platform for high-performance nucleic acid and protein analyses
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.
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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2008 (English)In: Nucleic Acids Research, ISSN 0305-1048, Vol. 36, no 8, e45- p.Article in journal (Refereed) Published
Abstract [en]

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.

Place, publisher, year, edition, pages
2008. Vol. 36, no 8, e45- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96777DOI: 10.1093/nar/gkn106ISI: 000255759600001PubMedID: 18346972OAI: oai:DiVA.org:uu-96777DiVA: diva2:171463
Available from: 2008-02-28 Created: 2008-02-28 Last updated: 2009-11-04Bibliographically approved
In thesis
1. Biomolecular Analysis by Dual-Tag Microarrays and Single Molecule Amplification
Open this publication in new window or tab >>Biomolecular Analysis by Dual-Tag Microarrays and Single Molecule Amplification
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges.

The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 315
Keyword
Molecular medicine, Microarray, proximity ligation, Padlock probe, Rolling circle amplification, Splicing, single molecule, Molekylärmedicin
Identifiers
urn:nbn:se:uu:diva-8475 (URN)978-91-554-7101-9 (ISBN)
Public defence
2008-03-20, Rudbeckssalen, Rudbeckslaboratoriet, Daghammarsköldsväg 20, Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-02-28 Created: 2008-02-28Bibliographically approved

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