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Biomolecular Analysis by Dual-Tag Microarrays and Single Molecule Amplification

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.

Identiferoai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-8475
Date January 2008
CreatorsEricsson, Olle
PublisherUppsala universitet, Institutionen för genetik och patologi, Uppsala : Acta Universitatis Upsaliensis
Source SetsDiVA Archive at Upsalla University
LanguageEnglish
Detected LanguageEnglish
TypeDoctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text
Formatapplication/pdf
Rightsinfo:eu-repo/semantics/openAccess
RelationDigital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, 1651-6206 ; 315

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