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Visual DNA: Identification of DNA sequence variations by bead trapping
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0002-2207-7370
KTH, School of Biotechnology (BIO), Nano Biotechnology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
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2007 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 90, 741-745 p.Article in journal (Refereed) Published
Abstract [en]

In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.

Place, publisher, year, edition, pages
2007. Vol. 90, 741-745 p.
Keyword [en]
imaging techniques; medical imaging; magnetics; microspheres; oligonucleotide array; sequence analysis; signal amplification assay
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11218DOI: 10.1016/j.ygeno.2007.07.014ISI: 000251707500010OAI: oai:DiVA.org:kth-11218DiVA: diva2:242207
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Novel diagnostic microarray assay formats towards comprehensive on-site analysis
Open this publication in new window or tab >>Novel diagnostic microarray assay formats towards comprehensive on-site analysis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 90 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:18
Keyword
magnetic microbeads, gold nanobeads, microarrays, lateral flow, on-site diagnosis, SNPs, pestivirus, autoimmunity, contagious bovine pleuropneumonia, comsol multiphysics, finite element method
National Category
Microbiology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-11221 (URN)978-91-7415-416-0 (ISBN)
Public defence
2009-10-23, FR4 Oskar Klein, AlbaNova Universitetscentrum, Roslagstullsbacken, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-11-23Bibliographically approved
2. 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.

Publisher
53 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:4
Keyword
array, sequence capture, genotyping, trinucleotide threading, sequencing, massively parallel sequencing, single molecule sequencing, Visual DNA, p53, single nucleotide polymorphism, biomarker
National Category
Genetics
Identifiers
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)
Opponent
Supervisors
Available from: 2010-04-19 Created: 2010-04-16 Last updated: 2010-08-26

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