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Multiplex PCR amplification using Extractor probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Nilsson)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Manipulation of a plurality of DNA molecules in parallel offers several advantages over simplex reactions. This paper reports on a new version of Selector probes with easier design procedure and the possibility to achieve higher ROI content within the targeted fragments. To demonstrate its utility we applied it for copy number variance detection and simultaneous amplification of 64 genomic fragments.

Keyword [en]
PCR, sequencing, genetics, CNV
National Category
Medical Genetics
Research subject
URN: urn:nbn:se:uu:diva-121532OAI: oai:DiVA.org:uu-121532DiVA: diva2:305662
Available from: 2010-03-24 Created: 2010-03-24 Last updated: 2010-03-25
In thesis
1. Microfluidic and Molecular Tools for Genetic Analyses
Open this publication in new window or tab >>Microfluidic and Molecular Tools for Genetic Analyses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments.

The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences.

A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping.

The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 36 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 546
microfluidics, PCR, sequencing, cancer genetics
National Category
Medical Genetics
Research subject
Medical Genetics
urn:nbn:se:uu:diva-121536 (URN)978-91-554-7765-3 (ISBN)
Public defence
2010-05-05, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Available from: 2010-04-14 Created: 2010-03-24 Last updated: 2010-04-14Bibliographically approved

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Johansson, Henrik
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