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MLGA--a rapid and cost-efficient assay for gene copy-number 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.
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 Immunology, Genetics and Pathology, Medical Genetics.
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2007 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 17, e115- p.Article in journal (Refereed) Published
Abstract [en]

Structural variation is an important cause of genetic variation. Whole genome analysis techniques can efficiently identify copy-number variable regions but there is a need for targeted methods, to verify and accurately size variable regions, and to diagnose large sample cohorts. We have developed a technique based on multiplex amplification of size-coded selectively circularized genomic fragments, which is robust, cheaper and more rapid than current multiplex targeted copy-number assays.

Place, publisher, year, edition, pages
2007. Vol. 35, no 17, e115- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-13111DOI: 10.1093/nar/gkm651ISI: 000250683500008PubMedID: 17823203OAI: oai:DiVA.org:uu-13111DiVA: diva2:40881
Available from: 2008-01-21 Created: 2008-01-21 Last updated: 2017-12-11Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 543
Keyword
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
Identifiers
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)
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Available from: 2010-04-14 Created: 2010-03-23 Last updated: 2010-04-16

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Stenberg, JohanThuresson, Ann-CharlotteBondeson, Marie-LouiseNilsson, Mats

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