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Single molecule analysis of combinatorial splicing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Landegren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Nilsson)
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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2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 16, e163- p.Article in journal (Refereed) Published
Abstract [en]

Alternative splicing forms diverse mRNA isoform populations from a single ancestral pre-mRNA and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns into a library of binary strings of nucleic acid tags, encoding the exon composition of transcripts. The transcript abundance is registered by counts of individual molecules and individual exon inclusion patterns are represented as strings of binary data.

The technique is illustrated by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits different genes to be analyzed in parallel and will be valuable for elucidating the complex effects of combinatorial splicing.

Place, publisher, year, edition, pages
2010. Vol. 38, no 16, e163- p.
Keyword [en]
Splicing, Combinatorial, RCA, Single molecule, Amplified Single Molecule Detection
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-96780DOI: 10.1093/nar/gkq581ISI: 000281720500003PubMedID: 20587504OAI: oai:DiVA.org:uu-96780DiVA: diva2:171466
Available from: 2010-03-11 Created: 2008-02-28 Last updated: 2017-12-14Bibliographically 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
2. Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation
Open this publication in new window or tab >>Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples.

We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I).

Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II).

Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III).

This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 39 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 532
Keyword
ligase, proximity ligation, gastric cancer, glycosylation, alternative splicing, avian influenza, padlock probe
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-120016 (URN)978-91-554-7743-1 (ISBN)
Public defence
2010-04-23, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds Väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-04-01 Created: 2010-03-04 Last updated: 2010-04-01Bibliographically approved

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Conze, TimAkusjärvi, GöranLandegren, UlfNilsson, Mats

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