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Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation
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, Molecular tools. (Landegren)
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. , p. 39
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 532
Keywords [en]
ligase, proximity ligation, gastric cancer, glycosylation, alternative splicing, avian influenza, padlock probe
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-120016ISBN: 978-91-554-7743-1 (print)OAI: oai:DiVA.org:uu-120016DiVA, id: diva2:302593
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: 2018-01-12Bibliographically approved
List of papers
1. Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
Open this publication in new window or tab >>Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
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2008 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 5, p. 1747-1751Article in journal (Refereed) Published
Abstract [en]

A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-16412 (URN)10.1128/JCM.02292-07 (DOI)000255678200027 ()18353937 (PubMedID)
Available from: 2008-05-22 Created: 2008-05-22 Last updated: 2017-12-08Bibliographically approved
2. Single molecule analysis of combinatorial splicing
Open this publication in new window or tab >>Single molecule analysis of combinatorial splicing
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2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 16, p. e163-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.

Keywords
Splicing, Combinatorial, RCA, Single molecule, Amplified Single Molecule Detection
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-96780 (URN)10.1093/nar/gkq581 (DOI)000281720500003 ()20587504 (PubMedID)
Available from: 2010-03-11 Created: 2008-02-28 Last updated: 2017-12-14Bibliographically approved
3. MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
Open this publication in new window or tab >>MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
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2010 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 2, p. 199-206Article in journal (Refereed) Published
Abstract [en]

Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-112103 (URN)10.1093/glycob/cwp161 (DOI)000273226500007 ()19815850 (PubMedID)
Available from: 2010-01-08 Created: 2010-01-08 Last updated: 2017-12-12Bibliographically approved

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