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1.
Genome Res ; 30(6): 898-909, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32540955

RESUMEN

Long-range sequencing information is required for haplotype phasing, de novo assembly, and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirements. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-seq) technology, which enables a low-cost, high-accuracy, and high-throughput short-read second-generation sequencer to generate over 100 kb of long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcoded linked-reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate megabase-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.


Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Biología Computacional/métodos , Código de Barras del ADN Taxonómico/métodos , Variación Genética , Genoma Humano , Genómica/métodos , Antígenos HLA/genética , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Flujo de Trabajo
3.
Acc Chem Res ; 47(6): 1750-8, 2014 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-24803094

RESUMEN

CONSPECTUS: DNA based nanotechnology provides a basis for high-resolution fabrication of objects almost without physical size limitations. However, the pathway to large-scale production of large objects is currently unclear. Operationally, one method forward is to use high information content, large building blocks, which can be generated with high yield and reproducibility. Although flat DNA origami naturally invites comparison to pixels in zero, one, and two dimensions and voxels in three dimensions and has provided an excellent mechanism for generating blocks of significant size and complexity and a multitude of shapes, the field is young enough that a single "brick" has not become the standard platform used by the majority of researchers in the field. In this Account, we highlight factors we considered that led to our adoption of a cross-shaped, non-space-filling origami species, designed by Dr. Liu of the Seeman laboratory, as the building block ideal for use in the fabrication of finite one-dimensional arrays. Three approaches that can be employed for uniquely coding origami-origami linkages are presented. Such coding not only provides the energetics for tethering the species but also uniquely designates the relative orientation of the origami building blocks. The strength of the coding approach implemented in our laboratory is demonstrated using examples of oligomers ranging from finite multimers composed of four, six, and eight origami structures to semi-infinite polymers (100mers). Two approaches to finite array design and the series of assembly steps that each requires are discussed. The process of AFM observation for array characterization is presented as a critical case study. For these soft species, the array images do not simply present the solution phase geometry projected onto a two-dimensional surface. There are additional perturbations associated with fluidic forces associated with sample preparation. At this time, reconstruction of the "true" or average solution structures for blocks is more readily achieved using computer models than using direct imaging methods. The development of scalable 1D-origami arrays composed of uniquely addressable components is a logical, if not necessary, step in the evolution of higher order fully addressable structures. Our research into the fabrication of arrays has led us to generate a listing of several important areas of future endeavor. Of high importance is the re-enforcement of the mechanical properties of the building blocks and the organization of multiple arrays on a surface of technological importance. While addressing this short list of barriers to progress will prove challenging, coherent development along each of these lines of inquiry will accelerate the appearance of commercial scale molecular manufacturing.


Asunto(s)
ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico
4.
Front Phys ; 62018.
Artículo en Inglés | MEDLINE | ID: mdl-31667164

RESUMEN

Macromolecular crowding plays a principal role in a wide range of biological processes including gene expression, chromosomal compaction, and viral infection. However, the impact that crowding has on the dynamics of nucleic acids remains a topic of debate. To address this problem, we use single-molecule fluorescence microscopy and custom particle-tracking algorithms to investigate the impact of varying macromolecular crowding conditions on the transport and conformational dynamics of large DNA molecules. Specifically, we measure the mean-squared center-of-mass displacements, as well as the conformational size, shape, and fluctuations, of individual 115 kbp DNA molecules diffusing through various in vitro solutions of crowding polymers. We determine the role of crowder structure and concentration, as well as ionic conditions, on the diffusion and configurational dynamics of DNA. We find that branched, compact crowders (10 kDa PEG, 420 kDa Ficoll) drive DNA to compact, whereas linear, flexible crowders (10, 500 kDa dextran) cause DNA to elongate. Interestingly, the extent to which DNA mobility is reduced by increasing crowder concentrations appears largely insensitive to crowder structure (branched vs. linear), despite the highly different configurations DNA assumes in each case. We also characterize the role of ionic conditions on crowding-induced DNA dynamics. We show that both DNA diffusion and conformational size exhibit an emergent non-monotonic dependence on salt concentration that is not seen in the absence of crowders.

5.
Sci Rep ; 8(1): 17817, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30546025

RESUMEN

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus that can induce both lytic and latent infections in various insect cell lines. During latent infection, several microRNAs (miRNAs) are produced from persistency-associated gene 1 (pag1) as the only detectable HzNV-1 transcript. Previous studies have shown that the pag1 gene suppresses the immediate-early gene hhi1 and promotes host switching into a latent infection via miRNAs derived from pag1. Although other functions of the miRNAs derived from pag1 have not yet been elucidated, several studies have suggested that miRNAs encoded from latency-associated genes can regulate histone-associated enzymes. Because pag1 is a noncoding transcript, it potentially regulates host chromatin structure through miRNAs upon infection. Nevertheless, the exact mechanism by which pag1 alters viral infections remains unknown. In this study, we found that the pag1-encoded miRNA miR-420 suppresses expression of the histone modification-associated enzyme su(var)3-9. Therefore, this miRNA causes histone modification to promote HzNV-1 infection. These results suggest that HzNV-1 may directly influence epigenetic regulation in host cells through interactions with pag1 miRNAs to promote lytic infection. This study provides us with a better understanding of both the HzNV-1 infection pathway and the relationship between viral miRNAs and epigenetic regulation.


Asunto(s)
Epigénesis Genética , Regulación Viral de la Expresión Génica , Histonas/metabolismo , Proteínas de Insectos/metabolismo , MicroARNs/biosíntesis , Nucleopoliedrovirus/fisiología , ARN Viral/biosíntesis , Spodoptera , Animales , Metilación , Células Sf9 , Spodoptera/metabolismo , Spodoptera/virología , Proteínas Virales/metabolismo
6.
Sci Rep ; 7: 41255, 2017 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-28112264

RESUMEN

The western honeybee (Apis mellifera) is essential for the global economy due to its important role in ecosystems and agriculture as a pollinator of numerous flowering plants and crops. Pesticide abuse has greatly impacted honeybees and caused tremendous loss of honeybee colonies worldwide. The reasons for colony loss remain unclear, but involvement of pesticides and pathogen-pesticide interactions has been hypothesized. Histone deacetylase inhibitors (HDACis) inhibit the activity of histone acetylase, which causes the hyperacetylation of histone cores and influences gene expression. In this study, sodium butyrate, an HDACi, was used as a dietary supplement for honeybees; after treatment, gene expression profiles were analyzed using quantitative PCR. The results showed that sodium butyrate up-regulated genes involved in anti-pathogen and detoxification pathways. The bioassay results showed that honeybees treated with sodium butyrate were more tolerant to imidacloprid. Additionally, sodium butyrate strengthened the immune response of honeybees to invasions of Nosema ceranae and viral infections. We also performed a bioassay in which honeybees were exposed to pesticides and pathogens. Our results provide additional data regarding the mechanism by which honeybees react to stress and the potential application of HDACis in beekeeping.


Asunto(s)
Abejas/efectos de los fármacos , Abejas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Transducción de Señal/genética , Acetilación/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Abejas/inmunología , Abejas/microbiología , Ácido Butírico/farmacología , Caspasa 3/metabolismo , Inactivación Metabólica/efectos de los fármacos , Inactivación Metabólica/genética , Microsporidiosis/genética , Microsporidiosis/patología , Neonicotinoides/farmacología , Nitrocompuestos/farmacología , Nosema/efectos de los fármacos , Nosema/fisiología , Transducción de Señal/efectos de los fármacos
7.
IEEE Trans Nanobioscience ; 12(2): 93-7, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23694695

RESUMEN

Surface-enhanced Raman scattering is used to study the Raman spectra and peak shifts the thrombin-binding aptamer (TBA) on substrates having two different geometries; one with a single stranded sequence and one with double stranded sequence. The Raman signals of the deoxyribonucleic acids on both substrates are enhanced and specific peaks of bases are identified. These results are highly reproducible and have promising applications in low cost nucleic acid detection.


Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Oro/química , Nanopartículas del Metal/química , Potasio/química , Espectrometría Raman
8.
IEEE Trans Nanobioscience ; 12(4): 282-8, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25140361

RESUMEN

Herein, we investigate the migration distance of quantum-dot-functionalized complexes in electrophoresis. The quantitative study of these moving particles in an electrophoretic environment is modeled using an extended Smoluchowski equation. An extended Smoluchowski equation is proposed to addressed the D(m) to Ln(N) plot slope variation issue present in previous work and agreement between experiment and theory is found. The procedures underlying this work then discusses the potential of using agarose electrophoresis as a mean of monitoring the composition of nano-complexes consisting of quantum dots functionalized with differing numbers of DNA molecules.


Asunto(s)
ADN de Cadena Simple/química , Electroforesis en Gel de Agar/métodos , Puntos Cuánticos/química , ADN de Cadena Simple/metabolismo , Puntos Cuánticos/metabolismo
9.
IEEE Trans Nanobioscience ; 12(3): 233-8, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23722479

RESUMEN

Signature molecules derived from Listeria monocytogenes, Bacillus thuringiensis, and Salmonella Typhimurium were detected directly on food substrates (mega) by coupling molecular beacon technology utilizing fluorescent resonance energy transfer (FRET), luminescent nanoscale semiconductor quantum dots, and nanoscale quenchers. We designed target DNA sequences for detecting hlyA, Bt cry1Ac, and invA genes from L. monocytogenes, B. thuringiensis and Salmonella Typhimurium, respectively, and prepared molecular beacons for specific targets for use in real-time monitoring. We successfully detected increased fluorescence in the presence of signature molecules at molecular beacon (MB) concentrations from 1.17 nM to 40 nM, depending upon system tested in (water, milk or plant leaves), respective target (hlyA, Bt cry1Ac, or invA) and genomic DNA target concentration (50-800 ng). We were able to detect bacterial genomic DNA derived from L. monocytogenes and Salmonella sp. in a food system, 2% milk ( > 20% of total volume). Furthermore, we infiltrated the Bt cry1Ac beacon in the presence of genomic DNA extracted from B. thuringiensis into Arabidopsis thaliana leaves and observed increased fluorescence in the presence of the target, indicating the ability to use these beacons in a plant system.


Asunto(s)
ADN Bacteriano/análisis , Enfermedades Transmitidas por los Alimentos/microbiología , Técnicas de Sonda Molecular , Puntos Cuánticos , Animales , Arabidopsis/microbiología , Bacillus thuringiensis/genética , Bacillus thuringiensis/aislamiento & purificación , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Microbiología de Alimentos , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Leche/microbiología , Tipificación Molecular , Nanomedicina , Hojas de la Planta/microbiología , Salmonella/genética , Salmonella/aislamiento & purificación , Transgenes
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