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Real-time monitoring of rolling-circle amplification using a modified molecular beacon design
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.
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2002 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 14, e66- p.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.

Place, publisher, year, edition, pages
2002. Vol. 30, no 14, e66- p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93438DOI: 10.1093/nar/gnf065PubMedID: 12136114OAI: oai:DiVA.org:uu-93438DiVA: diva2:166912
Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Selector Technology: For Multiplex DNA Analysis
Open this publication in new window or tab >>Selector Technology: For Multiplex DNA Analysis
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR.

We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR.

A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 65
Keyword
Genetic engineering, DNA technology, Genteknik
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-5921 (URN)91-554-6330-4 (ISBN)
Public defence
2005-10-07, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2009-10-14Bibliographically approved
2. 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

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