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Proximity Ligation as a Universal Protein Detection Tool
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today.

This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously.

Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 51
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1222
Keywords [en]
Molecular medicine, Proximity ligation, proteomics, aptamer, antibody, molecular beacon, rolling circle amplification
Keywords [sv]
Molekylärmedicin
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-3294ISBN: 91-554-5521-2 (print)OAI: oai:DiVA.org:uu-3294DiVA, id: diva2:162333
Public defence
2003-02-28, The Rudbeck hall, Uppsala, 09:15
Opponent
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Protein detection using proximity-dependent DNA ligation assays
Open this publication in new window or tab >>Protein detection using proximity-dependent DNA ligation assays
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2002 (English)In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 20, no 5, p. 473-477Article in journal (Refereed) Published
Abstract [en]

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Keywords
Animals, Base Sequence, Chemistry; Clinical/*methods, DNA/*metabolism, Dose-Response Relationship; Drug, Enzyme-Linked Immunosorbent Assay, Humans, Molecular Sequence Data, Oligonucleotides/*metabolism, Platelet-Derived Growth Factor/*analysis/pharmacology, Proteins/*analysis, Sensitivity and Specificity, Thrombin/pharmacology, Time Factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-90115 (URN)10.1038/nbt0502-473 (DOI)11981560 (PubMedID)
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2017-12-14Bibliographically approved
2. Cytokine detection by antibody-based proximity ligation
Open this publication in new window or tab >>Cytokine detection by antibody-based proximity ligation
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90116 (URN)
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2010-01-13Bibliographically approved
3. Real-time monitoring of rolling-circle amplification using a modified molecular beacon design
Open this publication in new window or tab >>Real-time monitoring of rolling-circle amplification using a modified molecular beacon design
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2002 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 14, p. e66-Article in journal (Refereed) Published
Abstract [en]

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93438 (URN)10.1093/nar/gnf065 (DOI)12136114 (PubMedID)
Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-12-14Bibliographically approved
4. In situ detection of proteins by proximity ligation
Open this publication in new window or tab >>In situ detection of proteins by proximity ligation
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90118 (URN)
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2010-01-13Bibliographically approved

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