13 research outputs found
Competitive-Binding Based Optical DNA Mapping - From Bacterial Plasmids to the Human Genome
Significant advances within the field of DNA sequencing have allowed us to study DNA at a level of detail that was previously impossible. However, dynamic genomic regions with a high degree of structural variations, while being linked to disease in humans and increased resistance to antibiotics, are still challenging to characterize. Furthermore, DNA sequencing for bacterial diagnostics and detection of resistance genes is presently hampered by the excessive lead times associated with the overall complexity of the applied methods.This Thesis describes the development of novel assays based on optical DNA mapping, which, although studying DNA at a lower resolution, is capable of rapid processing of significantly larger DNA fragments compared to sequencing. The fluorescent labeling in the assays presented here relies on competitive DNA binding between the emissive YOYO-1 and the sequence-specific, non-emissive, netropsin. The labeled DNA is then stretched in nanofluidic channels and imaged using fluorescence microscopy, enabling extraction of coarse-grained sequence information from ultralong DNA molecules at the single-molecule level.The results demonstrate how competitive binding-based optical DNA mapping can be used to characterize and trace bacterial DNA, responsible for the spread of antibiotic resistance. The mapped bacterial DNA can also be used to identify bacterial species in complex mixtures and directly from clinical samples. Additionally, so-called long-range sequence information of the human genome can be obtained, with possible future applications including detection of disease-related structural variations and epigenetic profiling
DNA in Nanochannels - Theory and Applications
Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is relatively easy to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides kilobase pair resolution of the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, rather than recent applications in human genetics, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with the chain ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology and biophysics
Identification of pathogenic bacteria in complex samples using a smartphone based fluorescence microscope
Diagnostics based on fluorescence imaging of biomolecules is typically performed in well-equipped laboratories and is in general not suitable for remote and resource limited settings. Here we demonstrate the development of a compact, lightweight and cost-effective smartphone-based fluorescence microscope, capable of detecting signals from fluorescently labeled bacteria. By optimizing a peptide nucleic acid (PNA) based fluorescence in situ hybridization (FISH) assay, we demonstrate the use of the smartphone-based microscope for rapid identification of pathogenic bacteria. We evaluated the use of both a general nucleic acid stain as well as species-specific PNA probes and demonstrated that the mobile platform can detect bacteria with a sensitivity comparable to that of a conventional fluorescence microscope. The PNA-based FISH assay, in combination with the smartphone-based fluorescence microscope, allowed us to qualitatively analyze pathogenic bacteria in contaminated powdered infant formula (PIF) at initial concentrations prior to cultivation as low as 10 CFU per 30 g of PIF. Importantly, the detection can be done directly on the smartphone screen, without the need for additional image analysis. The assay should be straightforward to adapt for bacterial identification also in clinical samples. The cost-effectiveness, field-portability and simplicity of this platform will create various opportunities for its use in resource limited settings and point-of-care offices, opening up a myriad of additional applications based on other fluorescence-based diagnostic assays
Strain-level bacterial typing directly from patient samples using optical DNA mapping
For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management
Optical Mapping of Bacterial Plasmids
Bacteria that have acquired resistance to antibiotics represent one of the largest threats to human health and modern health care. The genes encoding resistance are frequently spread via the transfer of plasmids, which are circular double stranded DNA molecules separate from the chromosomal DNA of the bacteria. The increasing prevalence of resistant bacteria, in combination with the absence of new major discoveries of antimicrobial drugs, means that health care could soon be entering a “post-antibiotic er". Besides developing new drugs, methods capable of rapidly detecting bacteria that have acquired resistance to antibiotics are of paramount importance for clinical point of care applications.In this Thesis, the development of an assay for rapid plasmid characterization is described. The method is based on optical DNA mapping using competitive binding of the fluorophore YOYO-1 and the sequence-specific, non-fluorescent, molecule netropsin, to DNA. The fluorescently labeled DNA is stretched in nanofluidic channels and imaged using fluorescence microscopy, enabling coarse-grained sequence information to be read from the intact plasmid at the single plasmid level. Additionally, an approach for gene detection on individual plasmids is described, combining the CRISPR/Cas9 system with optical DNA mapping.The results demonstrate how the assay can be used to obtain the number of different plasmids in a sample, the size of each plasmid, an optical barcode for tracing and identification, as well as information about which plasmid that carries a specific (resistance) gene. Overall, the assay shows great potential as a first step of plasmid characterization in point of care diagnostics
Optical DNA mapping in nanofluidic devices: principles and applications
Optical DNA mapping has over the last decade emerged as a very powerful tool for obtaining long range sequence information from single DNA molecules. In optical DNA mapping, intact large single DNA molecules are labeled, stretched out, and imaged using a fluorescence microscope. This means that sequence information ranging over hundreds of kilobasepairs (kbp) can be obtained in one single image. Nanochannels offer homogeneous and efficient stretching of DNA that is crucial to maximize the information that can be obtained from optical DNA maps. In this review, we highlight progress in the field of optical DNA mapping in nanochannels. We discuss the different protocols for sequence specific labeling and divide them into two main categories, enzymatic labeling and affinity-based labeling. Examples are highlighted where optical DNA mapping is used to gain information on length scales that would be inaccessible with traditional techniques. Enzymatic labeling has been commercialized and is mainly used in human genetics and assembly of complex genomes, while the affinity-based methods have primarily been applied in bacteriology, for example for rapid analysis of plasmids encoding antibiotic resistance. Next, we highlight how the design of nanofluidic channels can been altered in order to obtain the desired information and discuss how recent advances in the field make it possible to retrieve information beyond DNA sequence. In the outlook section, we discuss future directions of optical DNA mapping, such as fully integrated devices and portable microscopes
Developing an optical DNA mapping toolbox to identify chromosomal translocations in acute myeloid leukemia
We present a method based on CRISPR/Cas9 technology and optical DNA mapping (ODM) for clinical diagnosis of chromosomal translocations in Acute Myeloid Leukemia. CRISPR/Cas9 is used to cut-out genomic regions of interest, which are then differentially stained, stretched inside nanochannels and imaged. Sequence-dependent intensity pattern of stretched molecules enable studying linear association of genomic regions and pinpoint structural variations. Our method can reveal DNA structural details on length-scales that are difficult to address by state-of-the-art molecular diagnostic approaches. This manuscript reports proof-of-concept results for CRISPR/Cas9 excision of a fragment in the E.coli genome and high-fidelity mapping of the human genome
Enantioselective Cyclization of Photochromic Dithienylethenes Bound to DNA
Guiding light: Enantioselectivity is obtained for the photocyclization of a photochromic dithienylethene when isomerization is carried out in the presence of DNA (see scheme). Copyright \ua9 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Enantioselective Cyclization of Photochromic Dithienylethenes Bound to DNA
Guiding light: Enantioselectivity is obtained for the photocyclization of a photochromic dithienylethene when isomerization is carried out in the presence of DNA (see scheme). Copyright \ua9 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Detailed characterization of plasmids carrying resistance genes using optical DNA mapping
We present an assay, based on optical DNA mapping in nanochannels that is capable of characterizing the plasmid content of bacterial isolates resistant to antibiotics in a fast an detailed way. In a single experiment we determine the number of different plasmids in each sample, their size, as well as a barcode that can be used for plasmid identification and tracing. In addition we demonstrate that we can identify resistance genes on individual plasmids using CRISPR/Cas9. We foresee that the assay can be a useful tool all the way from fundamental plasmid biology to diagnostics and surveillance of resistant infections