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Advances in DNA Detection
KTH, School of Biotechnology (BIO), Gene Technology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice.

The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work.

The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model, which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , ix, 56 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:4
Keyword [en]
DNA detection, active filter paper, visual detection, throughput, fluorescence, superparamagnetic beads, rare DNA, Q-PCR, chemical fragmentation, DNA library preparation, massively parallel sequencing
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-143047ISBN: 978-91-7595-053-2 (print)OAI: oai:DiVA.org:kth-143047DiVA: diva2:705792
Public defence
2014-04-11, CMB Lecture hall, Berzelius väg 21, Karolinska Institute, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140318

Available from: 2014-03-18 Created: 2014-03-16 Last updated: 2014-03-18Bibliographically approved
List of papers
1. Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport
Open this publication in new window or tab >>Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport
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2012 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 7, 3311-3317 p.Article in journal (Refereed) Published
Abstract [en]

The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.

Keyword
Bioactive Paper, Visual Dna, Cellulose, Xyloglucan, Biosensors, Aptamers, Assay
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-95103 (URN)10.1021/ac300025v (DOI)000302829800039 ()22369042 (PubMedID)2-s2.0-84859392980 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20150629

Available from: 2012-05-21 Created: 2012-05-14 Last updated: 2017-12-07Bibliographically approved
2. Visual detection of DNA on paper chips
Open this publication in new window or tab >>Visual detection of DNA on paper chips
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2014 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 3, 1575-1582 p.Article in journal (Refereed) Published
Abstract [en]

On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.

Keyword
Analysis system, Capillary force, Cost-efficient, DNA analysis, Filter paper material, Nucleic acid assays, On-site detection, Visual detection
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-133777 (URN)10.1021/ac403196b (DOI)000331014800038 ()2-s2.0-84893634724 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research CouncilFormas
Note

QC 20140313. Updated from manuscript to article in journal.

Available from: 2013-11-11 Created: 2013-11-11 Last updated: 2017-12-06Bibliographically approved
3. Chemical fragmentation for massively parallel sequencing library preparation
Open this publication in new window or tab >>Chemical fragmentation for massively parallel sequencing library preparation
2013 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 168, no 1, 95-100 p.Article in journal (Refereed) Published
Abstract [en]

Fragmentation is essential in most library preparation protocols for use with massively parallel sequencing systems. Complexes that generate hydroxyl radicals, such as iron-EDTA, can be used to introduce random DNA cleavage. Here we describe a chemical fragmentation method that can be incorporated into library preparation protocols for next-generation sequencing workflows. This protocol has been validated by whole genome, amplicon and exome sequencing. Chemical fragmentation is a cost-effective alternative to current fragmentation methods that has no observable sequence bias and requires no instrumentation.

Keyword
Fragmentation, High-throughput sequencing, Library preparation
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-133269 (URN)10.1016/j.jbiotec.2013.08.020 (DOI)000325464300016 ()2-s2.0-84883827621 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research CouncilSwedish Foundation for Strategic Research
Note

QC 20131030

Available from: 2013-10-30 Created: 2013-10-29 Last updated: 2017-12-06Bibliographically approved
4. Nuclease-Assisted Suppression of Human DNA Background in Sepsis
Open this publication in new window or tab >>Nuclease-Assisted Suppression of Human DNA Background in Sepsis
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, e103610- p.Article in journal (Refereed) Published
Abstract [en]

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C(0)t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C(0)t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-143193 (URN)10.1371/journal.pone.0103610 (DOI)000340028800068 ()2-s2.0-84905054440 (Scopus ID)
Note

Updated from manuscript to article in journal.

QC 20140912

Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2017-12-05Bibliographically approved

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