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A single molecule array for digital targeted molecular analyses
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (M. Nilsson)
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. 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. (M. Nilsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (M. Nilsson)
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2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 1, e7- p.Article in journal (Refereed) Published
Abstract [en]

We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.

Place, publisher, year, edition, pages
England: Oxford University Press, , 2009. Vol. 37, no 1, e7- p.
Keyword [en]
padlock probe, RCA, single molecule detection, image analysis
National Category
Medical and Health Sciences
Research subject
Computerized Image Analysis; Molecular Biology
URN: urn:nbn:se:uu:diva-87970DOI: 10.1093/nar/gkn921ISI: 000262335700033OAI: oai:DiVA.org:uu-87970DiVA: diva2:134050
Available from: 2009-01-16 Created: 2009-01-16 Last updated: 2010-08-04Bibliographically approved
In thesis
1. Extracting Genomic Variations using Selector Technology
Open this publication in new window or tab >>Extracting Genomic Variations using Selector Technology
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development and use of a new class of molecular tools called Selector probes, and its potential for investigations of genetic variation. The Selector technology provides multiplex amplification of targeted DNA sequences with a high specificity, and an enrichment factor in the same order of magnitude as PCR. A common feature in this thesis work is to focus the analysis on DNA regions of interest. For example, this technique can be implemented in analysing candidate regions found by whole genome studies that need validation (global to local analysis), and applications requiring detection of rare alleles (common to rare allele), important in for example cancer samples.

An assay is presented that allows for fast and simple quantification of relative copy-number variations. The method was proven to be able to detect aneuploidy in chromosome 13, 18, 21 and X, with a resolution enough to distinguish between 4 and 5 copies. The method was successfully applied to solve a biological question regarding a copy-number variation, that explains the Ridge phenotype typical for the dog bread Rhodesian Ridgebacks. The Selector strategy was able to detect and map a tandem duplication with a size of 133 kb, which was characterized with base-pair resolution.

A readout platform that facilitates simultaneous digital quantitative analysis of a large numbers of biomolecules is further introduced. The work involves arraying amplified product from successful selection and decoding each molecule by hybridization of fluorophore labeled oligonucleotides.

Finally, a genome partitioning method which is applied upstream of next generation sequencing platforms is presented. It is shown that the method provides successful enrichment with 98 % coverage and 94 % specificity and high enrichment uniformity. The technique was applied for mutation analysis of 26 cancer-related genes in tumor cell-lines and tissue.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 46 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 543
Selector, Selector probe, Genetic variation, Resequencing, Targeted sequencing, copy-number variations, MLGA, Bioinformatics, Next generation sequencing, NGS, Amplified single molecule, ASM
National Category
Medical Genetics
Research subject
Genetics; Molecular Medicine
urn:nbn:se:uu:diva-121429 (URN)978-91-554-7761-5 (ISBN)
Public defence
2010-05-07, Rudbeckhall, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Available from: 2010-04-14 Created: 2010-03-23 Last updated: 2010-04-16

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