RESUMEN
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Imagen Individual de Molécula/métodos , Proteínas Virales/genética , Anticuerpos Antivirales/sangre , Secuencia de Bases , COVID-19/sangre , COVID-19/inmunología , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/normas , Prueba Serológica para COVID-19/normas , Ensayo de Inmunoadsorción Enzimática , Humanos , Sueros Inmunes/química , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Nasofaringe/virología , Poliproteínas/sangre , Poliproteínas/genética , ARN Viral/sangre , ARN Viral/genética , SARS-CoV-2/genética , Sensibilidad y Especificidad , Imagen Individual de Molécula/instrumentación , Proteínas Virales/sangreRESUMEN
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
RESUMEN
Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN de Plantas/genética , Genoma de Planta , Meiosis , Zea mays/genética , Mapeo Cromosómico , ADN de Plantas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Reparación del ADN por Recombinación , Secuencias Repetidas en Tándem , Zea mays/metabolismoRESUMEN
Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.
Asunto(s)
Agrobacterium tumefaciens/genética , Metilación de ADN/genética , ADN Bacteriano/genética , Genoma de Planta/genética , Arabidopsis/genética , Cromatina/genética , Epigenómica , Eucromatina/genética , Técnicas de Transferencia de Gen , Nucleosomas/genética , Motivos de Nucleótidos/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes.
Asunto(s)
Intercambio Genético/genética , Meiosis/genética , Recombinación Genética/genética , Cromatina/genética , Intercambio Genético/fisiología , ADN de Plantas/genética , Epigénesis Genética/genética , Genoma de Planta , Meiosis/fisiología , Recombinación Genética/fisiologíaRESUMEN
The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within â¼2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.
