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Nuclease-Assisted Suppression of Human DNA Background in Sepsis
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-8879-9245
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2014 (English)In: PLoS ONE, 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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 7, e103610- p.
National Category
Analytical Chemistry
URN: urn:nbn:se:kth:diva-143193DOI: 10.1371/journal.pone.0103610ISI: 000340028800068ScopusID: 2-s2.0-84905054440OAI: diva2:705806

Updated from manuscript to article in journal.

QC 20140912

Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2014-09-12Bibliographically approved
In thesis
1. Advances in DNA Detection
Open this publication in new window or tab >>Advances in DNA Detection
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.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:4
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
urn:nbn:se:kth:diva-143047 (URN)978-91-7595-053-2 (ISBN)
Public defence
2014-04-11, CMB Lecture hall, Berzelius väg 21, Karolinska Institute, Solna, 10:00 (English)
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20140318

Available from: 2014-03-18 Created: 2014-03-16 Last updated: 2014-03-18Bibliographically approved

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