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Visual DNA as a diagnostic tool
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
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2009 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 21, 3691-3695 p.Article in journal (Refereed) Published
Abstract [en]

We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin-coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE-based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand-held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off-the-shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point-of-care tests.

Place, publisher, year, edition, pages
2009. Vol. 30, no 21, 3691-3695 p.
Keyword [en]
Cystic fibrosis, Diagnostics, Mutation, PrAse, Visual DNA, CYSTIC-FIBROSIS GENE, IDENTIFICATION, TECHNOLOGIES
URN: urn:nbn:se:kth:diva-12401DOI: 10.1002/elps.200900273ISI: 000272126000006ScopusID: 2-s2.0-70649091306OAI: diva2:310836
qc 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2010-06-18Bibliographically approved
In thesis
1. Methods for Analyzing Genomes
Open this publication in new window or tab >>Methods for Analyzing Genomes
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions.

Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

53 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2010:4
array, sequence capture, genotyping, trinucleotide threading, sequencing, massively parallel sequencing, single molecule sequencing, Visual DNA, p53, single nucleotide polymorphism, biomarker
National Category
urn:nbn:se:kth:diva-12407 (URN)978-91-7415-596-9 (ISBN)
Public defence
2010-05-07, FD5, AlbaNova Universitetscentrum, Stockholm, 10:00 (English)
Available from: 2010-04-19 Created: 2010-04-16 Last updated: 2010-08-26

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