RESUMO
Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.
Assuntos
Tipagem de Bacteriófagos/métodos , Bacteriófagos/classificação , Bacteriófagos/genética , Código de Barras de DNA Taxonômico , Corantes Fluorescentes , Genoma Viral , Tipagem Molecular/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)RESUMO
We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Escherichia coli/classificação , Antibacterianos , Benzoxazóis , Ligação Competitiva , DNA Bacteriano/química , Escherichia coli/genética , Corantes Fluorescentes , Ligantes , Nanotecnologia , Netropsina , Compostos de QuinolínioRESUMO
Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
Assuntos
DNA/química , Técnicas Analíticas Microfluídicas , Composição de Bases , Benzoxazóis/química , Ligação Competitiva , Fluorescência , Corantes Fluorescentes/química , Netropsina/química , Compostos de Quinolínio/químicaRESUMO
Chromobacterium violaceum is a ß-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium.
Assuntos
Chromobacterium/genética , Anotação de Sequência Molecular , Plasmídeos/genética , Composição de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fases de Leitura Aberta , Análise de Sequência de DNARESUMO
The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.
Assuntos
Algoritmos , Mapeamento de Sequências Contíguas/métodos , Código de Barras de DNA Taxonômico , Benzoxazóis/metabolismo , Ligação Competitiva , Cromossomos/química , Simulação por Computador , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , Netropsina/metabolismo , Plasmídeos/genética , Estudo de Prova de Conceito , Compostos de Quinolínio/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de SequênciaRESUMO
DNA-protein interactions are at the core of the cellular machinery and single molecule methods have revolutionized the possibilities to study, and our understanding of these interactions on the molecular level. Nanofluidic channels have been extensively used for studying single DNA molecules during the last twelve years and in this review, we discuss how this experimental platform has been extended to studies of DNA-protein interactions. We first present how the design of the device can be tailored for the specific DNA-protein system studied and how the channels can be passivated to avoid non-specific binding of proteins. We then focus on describing the different kinds of DNA-interacting proteins that have been studied in nanofluidic devices, including proteins that compact DNA and proteins that form filaments on DNA. Our main objective is to highlight the diverse functionalities of DNA-protein systems that have been characterized using nanofluidic structures and hence demonstrate the versatility of these experimental tools. We finally discuss potential future directions studies of DNA-protein complexes in nanochannels might take, including specific DNA-protein systems that are difficult to analyze with traditional techniques, devices with increased complexity, and fully integrated lab-on-a-chip devices for analysis of material extracted from (single) cells.
Assuntos
DNA/química , DNA/metabolismo , Técnicas Analíticas Microfluídicas , Proteínas/química , Proteínas/metabolismo , Dispositivos Lab-On-A-Chip , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Ligação Proteica , Imagem Individual de Molécula/métodosRESUMO
This corrects the article DOI: 10.1038/srep30410.
RESUMO
Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.
Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos , Plasmídeos/genética , Sistemas CRISPR-Cas , Mapeamento Cromossômico , DNA Bacteriano/genética , Nanotecnologia , RNA Guia de Cinetoplastídeos/genética , Imagem Individual de MoléculaRESUMO
Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum ß-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance.
Assuntos
Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Plasmídeos/genética , Surtos de Doenças , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/metabolismo , Hospitais/estatística & dados numéricos , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo , Suécia/epidemiologiaRESUMO
The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.