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Homogeneous amplified single-molecule detection: Characterization of key parameters
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
2007 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 368, no 2, 230-238 p.Article in journal (Refereed) Published
Abstract [en]

We recently presented a method that enables single-molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target-specific padlock probe ligation, followed by rolling circle amplification (RCA), resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible using standard fluorescence microscopy by counting the number of generated RCPs in a sample pumped through a microfluidic channel. In this study, we demonstrate that confocal volume definition is crucial to achieve high-precision measurements in the microfluidic quantification (coefficient of variance typically 3%). We further demonstrate that complementary sequence motifs between RCPs is only a weak inducer of aggregates and that all detection sites of the RCPs are occupied at detection oligonucleotide concentrations greater than 5 nM if hybridized in the proper buffer conditions. Therefore, the signal/noise ratio is limited by the number of detection sites. By increasing the density of detection sites in the RCP by a factor of 1.9, we show that the optical signal/noise level can be increased from 42 to 75.

Place, publisher, year, edition, pages
2007. Vol. 368, no 2, 230-238 p.
Keyword [en]
Single-molecule detection, Padlock probes, Rolling circle amplification
National Category
Medical and Health Sciences Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-97692DOI: 10.1016/j.ab.2007.05.001ISI: 000248821700013PubMedID: 17572370OAI: oai:DiVA.org:uu-97692DiVA: diva2:172730
Available from: 2008-11-04 Created: 2008-11-04 Last updated: 2016-04-11Bibliographically approved
In thesis
1. Readout Strategies for Biomolecular Analyses
Open this publication in new window or tab >>Readout Strategies for Biomolecular Analyses
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 41 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 563
Keyword
Padlock probe, Selector probe, Rolling circle amplification, Single molecule detection, Amplified single molecule, Random array
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-9343 (URN)978-91-554-7320-4 (ISBN)
Public defence
2008-11-27, Rudbecksalen, Rudbeck Laboratory, C11, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2008-11-04 Created: 2008-11-04 Last updated: 2009-07-07Bibliographically approved

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