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Protein detection using proximity-dependent DNA ligation assays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Rudbeck Laboratory)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Rudbeck Laboratory)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Rudbeck Laboratory)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Rudbeck Laboratory)
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2002 (English)In: Nature Biotechnology, ISSN 1087-0156, Vol. 20, no 5, 473-477 p.Article 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.

Place, publisher, year, edition, pages
2002. Vol. 20, no 5, 473-477 p.
Keyword [en]
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: urn:nbn:se:uu:diva-90115DOI: 10.1038/nbt0502-473PubMedID: 11981560OAI: oai:DiVA.org:uu-90115DiVA: diva2:162329
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2013-05-31Bibliographically approved
In thesis
1. Proximity Ligation as a Universal Protein Detection Tool
Open this publication in new window or tab >>Proximity Ligation as a Universal Protein Detection Tool
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. 51 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1222
Keyword
Molecular medicine, Proximity ligation, proteomics, aptamer, antibody, molecular beacon, rolling circle amplification, Molekylärmedicin
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-3294 (URN)91-554-5521-2 (ISBN)
Public defence
2003-02-28, The Rudbeck hall, Uppsala, 09:15
Opponent
Available from: 2003-02-07 Created: 2003-02-07 Last updated: 2013-06-10Bibliographically approved
2. DNA Tools and Microfluidic Systems for Molecular Analysis
Open this publication in new window or tab >>DNA Tools and Microfluidic Systems for Molecular Analysis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.

Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected.

A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.

A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels.

I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction.

Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 161
Keyword
Molecular medicine, Proximity ligation, Microfluidics, Single molecule detection, Microarray, Bonding, Molekylärmedicin
Identifiers
urn:nbn:se:uu:diva-7079 (URN)91-554-6616-8 (ISBN)
Public defence
2006-09-21, Rudbecksalen, C11, Rudbecklaboratoriet, Uppsala, 09:15
Opponent
Supervisors
Available from: 2006-09-01 Created: 2006-09-01 Last updated: 2013-05-31Bibliographically approved

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