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Статті в журналах з теми "Optical replication mapping"

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Wang, Weitao, Kyle N. Klein, Karel Proesmans, Hongbo Yang, Claire Marchal, Xiaopeng Zhu, Tyler Borrman, et al. "Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication." Molecular Cell 81, no. 14 (July 2021): 2975–88. http://dx.doi.org/10.1016/j.molcel.2021.05.024.

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Lacroix, Joris, Sandrine Pélofy, Charline Blatché, Marie-Jeanne Pillaire, Sébastien Huet, Catherine Chapuis, Jean-Sébastien Hoffmann, and Aurélien Bancaud. "Analysis of DNA Replication by Optical Mapping in Nanochannels." Small 12, no. 43 (September 14, 2016): 5963–70. http://dx.doi.org/10.1002/smll.201503795.

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Rhind, Nick, Weitao Wang, Kyle Klein, Karel Proesmans, Hongbo Yang, Alex Hastie, Feng Yue, John Bechhoefer, Chen Chunlong, and David Gilbert. "Single‐Molecule Optical Replication Mapping (ORM) Suggests Human Replication Timing is Regulated by Stochastic Initiation." FASEB Journal 34, S1 (April 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.03352.

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Bayard, Quentin, Pierre Cordier, Camille Péneau, Sandrine Imbeaud, Theo Z. Hirsch, Victor Renault, Jean-Charles Nault, et al. "Structure, Dynamics, and Impact of Replication Stress–Induced Structural Variants in Hepatocellular Carcinoma." Cancer Research 82, no. 8 (February 25, 2022): 1470–81. http://dx.doi.org/10.1158/0008-5472.can-21-3665.

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Abstract Oncogene activation leads to replication stress and promotes genomic instability. Here we combine optical mapping and whole-genome sequencing (WGS) to explore in depth the nature of structural variants (SV) induced by replication stress in cyclin-activated hepatocellular carcinomas (CCN-HCC). In addition to classical tandem duplications, CCN-HCC displayed frequent intra-chromosomal and interchromosomal templated insertion cycles (TIC), likely resulting from template switching events. Template switching preferentially involves active topologically associated domains that are proximal to one another within the 3D genome. Template sizes depend on the type of cyclin activation and are coordinated within each TIC. Replication stress induced continuous accumulation of SVs during CCN-HCC progression, fostering the acquisition of new driver alterations and large-scale copy-number changes at TIC borders. Together, this analysis sheds light on the mechanisms, dynamics, and consequences of SV accumulation in tumors with oncogene-induced replication stress. Significance: Optical mapping and whole-genome sequencing integration unravels a unique signature of replication stress–induced structural variants that drive genomic evolution and the acquisition of driver events in CCN-HCC.
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Bayard, Quentin, Pierre Cordier, Camille Péneau, Sandrine Imbeaud, Theo Z. Hirsch, Victor Renault, Jean-Charles Nault, et al. "Abstract LB545: Structure, dynamics and consequences of replication stress-induced structural variants in hepatocellular carcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): LB545. http://dx.doi.org/10.1158/1538-7445.am2022-lb545.

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Abstract Oncogene activation leads to replication stress and promotes genomic instability. Here we combine optical mapping and whole genome sequencing to explore in-depth the nature of structural variants (SVs) induced by replication stress in cyclin-activated hepatocellular carcinomas (CCN-HCC). In addition to classical tandem duplications, CCN-HCC display frequent intra- and inter-chromosomal templated insertion cycles (TIC) likely resulting from template switching events. Template switching preferentially involves active topologically associated domains that are close in the 3D genome organization. Template sizes depend on the type of cyclin activation and are coordinated within each TIC. Replication stress induces continuous accumulation of SVs during CCN-HCC progression, fostering the acquisition of new driver alterations and large-scale copy-number changes at TIC borders. Together, this analysis sheds light on the mechanisms, dynamics and consequences of SV accumulation in tumors with oncogene-induced replication stress. Citation Format: Quentin Bayard, Pierre Cordier, Camille Péneau, Sandrine Imbeaud, Theo Z. Hirsch, Victor Renault, Jean-Charles Nault, Jean-Frédéric Blanc, Julien Calderaro, Chantal Desdouets, Jessica Zucman-Rossi, Eric Letouzé. Structure, dynamics and consequences of replication stress-induced structural variants in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB545.
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Brewer, Bonita J., Maitreya J. Dunham, and M. K. Raghuraman. "A unifying model that explains the origins of human inverted copy number variants." PLOS Genetics 20, no. 1 (January 4, 2024): e1011091. http://dx.doi.org/10.1371/journal.pgen.1011091.

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With the release of the telomere-to-telomere human genome sequence and the availability of both long-read sequencing and optical genome mapping techniques, the identification of copy number variants (CNVs) and other structural variants is providing new insights into human genetic disease. Different mechanisms have been proposed to account for the novel junctions in these complex architectures, including aberrant forms of DNA replication, non-allelic homologous recombination, and various pathways that repair DNA breaks. Here, we have focused on a set of structural variants that include an inverted segment and propose that they share a common initiating event: an inverted triplication with long, unstable palindromic junctions. The secondary rearrangement of these palindromes gives rise to the various forms of inverted structural variants. We postulate that this same mechanism (ODIRA: origin-dependent inverted-repeat amplification) that creates the inverted CNVs in inherited syndromes also generates the palindromes found in cancers.
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De Carli, Francesco, Nikita Menezes, Wahiba Berrabah, Valérie Barbe, Auguste Genovesio, and Olivier Hyrien. "High-Throughput Optical Mapping of Replicating DNA." Small Methods 2, no. 9 (August 6, 2018): 1800146. http://dx.doi.org/10.1002/smtd.201800146.

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Atkinson, Noah, Tyler A. Morhart, Garth Wells, Grace T. Flaman, Eric Petro, Stuart Read, Scott M. Rosendahl, Ian J. Burgess, and Sven Achenbach. "Microfabrication Process Development for a Polymer-Based Lab-on-Chip Concept Applied in Attenuated Total Reflection Fourier Transform Infrared Spectroelectrochemistry." Sensors 23, no. 14 (July 8, 2023): 6251. http://dx.doi.org/10.3390/s23146251.

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Micro electro-mechanical systems (MEMS) combining sensing and microfluidics functionalities, as are common in Lab-on-Chip (LoC) devices, are increasingly based on polymers. Benefits of polymers include tunable material properties, the possibility of surface functionalization, compatibility with many micro and nano patterning techniques, and optical transparency. Often, additional materials, such as metals, ceramics, or silicon, are needed for functional or auxiliary purposes, e.g., as electrodes. Hybrid patterning and integration of material composites require an increasing range of fabrication approaches, which must often be newly developed or at least adapted and optimized. Here, a microfabrication process concept is developed that allows one to implement attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and electrochemistry on an LoC device. It is designed to spatially resolve chemical sensitivity and selectivity, which are instrumental for the detection of chemical distributions, e.g., during on-flow chemical and biological reaction chemistry. The processing sequence involves (i) direct-write and soft-contact UV lithography in SUEX dry resist and replication in polydimethylsiloxane (PDMS) elastomers as the fluidic structure; (ii) surface functionalization of PDMS with oxygen plasma, 3-aminopropyl-triethoxysilane (APTES), and a UV-curable glue (NOA 73) for bonding the fluidic structure to the substrate; (iii) double-sided patterning of silicon nitride-coated silicon wafers serving as the ATR-FTIR-active internal reflection element (IRE) on one side and the electrode-covered substrate for microfluidics on the back side with lift-off and sputter-based patterning of gold electrodes; and (iv) a custom-designed active vacuum positioning and alignment setup. Fluidic channels of 100 μm height and 600 μm width in 5 mm thick PDMS were fabricated on 2” and 4” demonstrators. Electrochemistry on-chip functionality was demonstrated by cyclic voltammetry (CV) of redox reactions involving iron cyanides in different oxidation states. Further, ATR-FTIR measurements of laminar co-flows of H2O and D2O demonstrated the chemical mapping capabilities of the modular fabrication concept of the LoC devices.
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Fearns, Rachel, Mark E. Peeples, and Peter L. Collins. "Mapping the Transcription and Replication Promoters of Respiratory Syncytial Virus." Journal of Virology 76, no. 4 (February 15, 2002): 1663–72. http://dx.doi.org/10.1128/jvi.76.4.1663-1672.2002.

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ABSTRACT An important, unresolved issue in mononegavirus biology is whether or not transcription is initiated by the same promoter as RNA replication. In this study, residues important for respiratory syncytial virus (RSV) transcription and RNA replication were identified by subjecting the first 26 nucleotides of genome RNA to saturation mutagenesis. This analysis was performed using a genome analog that allowed transcription and RNA replication to be dissociated from each other and monitored as independent events in an intracellular assay. This analysis showed that nucleotides 3C, 5C, 8U, 9U, 10U, and 11U were important for transcription and RNA replication. Additional nucleotides (1U, 2G, 6U, and 7U) were important for RNA replication, but not transcription. At position 4, G versus C or U augmented transcription and decreased replication, showing that the naturally occurring assignments in the genomic (4G) and antigenomic (4U) promoters are optimal for transcription and RNA replication, respectively. These data show that RSV transcription and RNA replication each involve a cis-acting signal at the very 3" end of the genome. This signal appears to contain a minimum, common element that functions in both transcription and RNA replication, defined by those substitutions that had similar effects on the two processes. Apart from these common nucleotides, other positions were involved in RNA replication but not transcription or had different effects on the two processes. This indicates that the promoters for transcription and replication involve overlapping sets of nucleotides at the very 3" end of the genome and provides evidence that the nucleotide preferences for the two processes are not identical.
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Sung, Yen-Ling, Yuan-Si Tsai, Chu-Ling Chang, Chih-Kuan Chen, Chih-Hua Wang, and Yi-Chun Lin. "(Invited) Cardiac Engineering: Optical Mapping in Cellular Communication." ECS Meeting Abstracts MA2024-01, no. 33 (August 9, 2024): 1652. http://dx.doi.org/10.1149/ma2024-01331652mtgabs.

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Microfluidic platforms have emerged as valuable tools for replicating the adverse effects of chemotherapy drugs on cardiac and non-cardiac cells in vitro. However, these systems currently fall short of comprehensively capturing the intricate nature of human responses to chemotherapy, necessitating further validation to enhance model accuracy. While innovative chip systems provide insightful means for screening cardiac toxicity, they encounter challenges in addressing individual variations in drug responses and the influence of patient-specific factors. A novel cardiac chip incorporating intracellular electrophysiological techniques was developed to monitor acute hypoxia-induced responses in cardiac cells. Despite its promising potential, the model exhibits limitations inherent to its simplified two-dimensional microfluidic system, warranting additional validation and optimization efforts to enhance physiological relevance and facilitate applications in clinical translation. Moreover, previous research has proposed the formation of functional connections between human neurons and myocardial cells in a microfluidic chip. However, this study primarily focused on one-way connections between neurons and myocardial cells, overlooking the complexities of bidirectional neuro-muscular connections. Consequently, further research is imperative to deepen our understanding of the intricate interactions between neurons and myocardial cells. Optical mapping techniques assessed transmembrane voltage, mitochondrial activity, and calcium signals during electric pacing. These comprehensive assessments aim to advance our understanding of cardiac electrophysiology under drug exposure, providing crucial insights into the intricate responses of cardiac cells to chemotherapy. This research has significant implications for enhancing our understanding of cardiac safety in the context of chemotherapy.
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Дисертації з теми "Optical replication mapping"

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Wang, Weitao. "Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication." Electronic Thesis or Diss., Université Paris sciences et lettres, 2021. http://www.theses.fr/2021UPSLS048.

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La réplication de l'ADN est régulée par l'emplacement et le moment de l'initiation de la réplication. Par conséquent, beaucoup d'efforts ont été investis dans l'identification et l'analyse des sites d'initiation de la réplication dans les cellules humaines. Cependant, la nature hétérogène de la cinétique de réplication eucaryote et la faible efficacité de l'utilisation du site d'initiation individuelle chez les métazoaires a rendu difficile la cartographie de l'emplacement et du moment de l'initiation de la réplication dans les cellules humaines. Une solution potentielle au problème de la cartographie de la réplication humaine est l'analyse dans les molécules uniques. Cependant, les approches actuelles ne fournissent pas le débit requis pour les expériences à l'échelle du génome humaine. Pour relever ce défi, nous avons développé la cartographie de réplication optique (Optical Replicaiton Mapping - ORM), une approche de molécule unique à haut débit pour cartographier l'ADN nouvellement répliqué, et l'avons utilisée pour cartographier les événements d'initiation précoce dans les cellules humaines. La nature de molécule unique de nos données, et une couverture totale de plus de 2000 fois du génome humain sur 27 millions de fibres d'une longueur moyenne d'environ 300 kb, nous permettent d'identifier les sites d'initiation et leur probabilité d’initiation avec une grande confiance. En particulier, pour la première fois, nous sommes en mesure de mesurer à l'échelle du génome humain l'efficacité absolue de l'initiation de la réplication. Nous constatons que la distribution de l'initiation de la réplication humaine est cohérente avec l'initiation inefficace et stochastique de complexes d'initiation potentiels distribués de manière hétérogène enrichis en chromatine accessible. En particulier, nous constatons que les sites d'initiation de la réplication humaine ne sont pas limités à des origines de réplication bien définies, mais sont plutôt répartis sur de larges zones d'initiation constituées de nombreux sites d'initiation. De plus, nous ne trouvons aucune corrélation des événements d'initiation entre les zones d'initiation voisines. Bien que la plupart des événements d'initiation précoce se produisent dans les régions à réplication précoce du génome, un nombre significatif se produit dans les régions tardives. Le fait que les sites d'initiation dans les régions tardive aient une certaine probabilité d’initiation au début de la phase S suggère que la principale différence entre les événements d'initiation dans les régions à réplication précoce et tardive est leur probabilité intrinsèque d’initiation, et n’est pas due à une différence qualitative dans leur distribution de temps d’initiation. De plus, la modélisation de la cinétique de réplication démontre que la mesure de l'efficacité d’initiation de la zone d'initiation au début de la phase S suffit pour prédire le temps d’initiation moyen de ces zones tout au long de la phase S, ce qui suggère en outre que les différences entre les temps d’initiation des zones d'initiation précoce et tardive sont quantitatives plutôt que qualitatives. Ces observations sont cohérentes avec les modèles stochastiques de la régulation de l'initiation et suggèrent que la régulation stochastique de la cinétique de réplication est une caractéristique fondamentale de la réplication chez eucaryotes, conservée de la levure à l'homme
DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late replicating regions. The fact that initiation sites in typically late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans
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Saulebekova, Dalila. "Study of DNA replication program of the human genome by high–throughput single-molecule Optical Replication Mapping." Electronic Thesis or Diss., Sorbonne université, 2024. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2024SORUS185.pdf.

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Анотація:
La réplication de l'ADN est un processus cellulaire crucial, qui garantit que chaque division cellulaire aboutit à une duplication précise du génome, composé de plus de 6 milliards de paires de bases chez l'homme. Ce processus repose sur l'activation précise de milliers d'origines de réplication dans un ordre temporel défini, appelé programme Replication Timing (RT). Ce programme est étroitement lié à l'organisation de la chromatine et sa dérégulation peut conduire à l'instabilité du génome, à des mutations et au développement des maladies telles que le cancer. Cependant, la nature hétérogène et stochastique de l'activation des origines dans les cellules de mammifères pose des défis importants à notre compréhension de l'initiation de la réplication de l'ADN chez l'homme. L'organisation de la chromatine et la RT sont intimement liées à la fonction du génome. La protéine RIF1, conservée au cours de l'évolution, joue un rôle clé dans le contrôle de ces processus. Bien que l'importance de RIF1 dans la régulation de la chronologie de la réplication de l'ADN et de l'organisation de la chromatine soit bien étudiée, la question de savoir si RIF1 affecte la localisation et l'efficacité des sites d'initiation de la réplication n'a pas été élucidée. Dans ce travail, pour étudier l'impact détaillé de RIF1 sur l'initiation et la dynamique des origines de réplication, nous avons appliqué la cartographie optique de la réplication (ORM) - une approche à haut débit et à molécule unique récemment développée par notre équipe, qui combine la détection fluorescente des origines actives marquées in vivo sur de longues molécules d'ADN individuelles et leur cartographie optique sur le génome en utilisant la technologie Bionano Genomics. Nos résultats obtenus à partir de cellules HCT116 en phase S précoce comblent ce manque de recherche en démontrant que la déplétion de RIF1 entraîne un changement radical dans la localisation des origines et l'efficacité des tirs, montrant un tir plus homogène à travers le génome, et remettant en question les hypothèses antérieures sur la spécificité et l'efficacité des origines de réplication. Notamment, notre approche ORM améliorée a permis la découverte d'un grand nombre de nouvelles origines activées lors de la déplétion de RIF1, ainsi qu'une caractérisation détaillée des schémas de modification des histones associés. Nos résultats révèlent également les différences dans l'initiation des origines par rapport aux nouveaux états de la chromatine : nous avons observé que l'absence de RIF1 n'avait pas d'impact uniforme sur tous les états de la chromatine. Plus précisément, la déplétion de RIF1 augmente significativement l'initiation de la réplication dans le nouvel état B0, caractérisé par un enrichissement en H3K9me2 et H2A.Z et des préférences d'interactions neutres pour les compartiments A et B, et n'affecte que très peu l'hétérochromatine B4 qui se réplique tardivement. Pour avancer l'étude des régions de réplication tardive, nous avons amélioré la méthode ORM en améliorant l'efficacité du marquage, ce qui nous a permis de calculer un profil de directionnalité de la fourche de réplication (RFD) à haute résolution directement à partir des cellules asynchronisées. Les profils RFD ont révélé la dynamique d'initiation dans les régions tardives, y compris les sites fragiles communs (CFS), et ont confirmé l'activation d'origines de réplication tardives précédemment absentes.En combinant des approches multi-omiques et ORM, ce travail démontre pour la première fois que RIF1 ne modifie pas seulement la RT mais que son absence conduit également à l'activation de nouvelles origines, fournissant un support important pour comprendre davantage les mécanismes moléculaires gouvernant la réplication de l'ADN et l'organisation du génome
DNA replication is a crucial cellular process, ensuring that each cell division results in an accurate duplication of genome composed of > 6 billion base pairs in humans. This process relies on the precise activation of thousands of replication origins in a defined temporal order called Replication Timing (RT) program. This program is tightly linked to chromatin organization and its deregulation can lead to genome instability, mutations and development of diseases, such as cancer. However, heterogeneous and stochastic nature of origins activation in mammalian cells poses significant challenges to our understanding of DNA replication initiation in humans. Chromatin organization and RT are intricately linked to genome function, with theevolutionary conserved protein RIF1 playing a key role in controlling these processes. Although the importance of RIF1 in regulating the timing of DNA replication and the chromatin organization is well studied, whether RIF1 affects the location and efficiency of replication initiation sites remained unclear. In this work, to investigate the detailed impact of RIF1 on replication origins initiation and dynamics, we applied Optical Replication Mapping, (ORM) - a high-throughput, single-molecule approach recently developed by our team, that combines the fluorescent detection of in vivo labelled active origins over long individual DNA molecules and their optical mapping to the genome using Bionano Genomics technology. Our results obtained from early S-phase HCT116 cells addresses the research gap by demonstrating that RIF1 depletion let to a dramatic change in origin location and firing efficiency, showing a more homogeneous firing across the genome, and challenging previous assumptions about replication origin specificity and efficiency. Notably, our enhanced ORM approach enabled the discovery of large number of new origins activated upon the depletion of RIF1, alongside a detailed characterization of the associated histone modification patterns. Our results also uncover the differences in origins initiation in relation to the newly classified chromatin states: we observed that the absence of RIF1 did not uniformly impact all chromatin states. Specifically, RIF1 depletion significantly enhances the replication initiation in the newly characterized B0 state, characterized by enriched H3K9me2 and H2A.Z and neutral interaction preferences for A and B compartments, and minimally affects the late-replicating B4 heterochromatin. To advance the study of late replication regions, we have upgraded the ORM method by improving the labelling efficiency that allowed us to compute a high-resolution Replication Fork Directionality profile (RFD) directly from the asynchronized cells. RFD profiles revealed the initiation dynamics in late regions including Common Fragile Sites (CFSs) and confirmed that the activation of previously absent late replication origins. Through combination of multi-omics and ORM approaches, this work for the first time demonstrates that RIF1 not only alters RT but it's absence also leads to the activation of new origins, providing an important support for further dissecting the molecular mechanisms governing DNA replication and genome organization
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Menezes, Braganca Nikita. "Cartographie pangénomique à haut débit et en molécule unique de la réplication de l'ADN." Electronic Thesis or Diss., Paris Sciences et Lettres (ComUE), 2019. http://www.theses.fr/2019PSLEE040.

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Анотація:
La réplication de l'ADN est un processus vital qui assure la transmission l'information génétique aux cellules filles. Chez les eucaryotes, la réplication du génome s'effectue en utilisant de multiples origines de réplication. Chez les métazoaires, la cartographie de la réplication demeure difficile. Les cartographies pangénomiques des origines de réplication chez l’Homme réalisées à l'aide de techniques de séquençage, ne s’accordent que modérément. Une explication possible de ces incohérences est que ces approches utilisent de grandes populations cellulaires qui ne nous donne qu’une image moyenne de la réplication. Ainsi, pour mieux comprendre la réplication de l'ADN et accéder à cette variabilité inter-cellulaire, il est fondamental de développer des techniques en molécule unique telle que le peignage moléculaire. Cependant, cette dernière est réfractaire à l'automatisation et empêche l'analyse pangénomique de la réplication. Pour surmonter ces obstacles, nous avons ré-employé un dispositif de cartographie optique basé sur de la microfluidique, le système Bionano Genomics Irys, pour le High Throughput Optical MApping of Replicating DNA (HOMARD). Typiquement, pour un “run”, nous recueillons plus de 34 000 images et plus de 63 000 Mpb d'ADN. Nos nouveaux outils open source, qui ont nécessité l'adaptation du logiciel propriétaire fourni, nous permettent de visualiser simultanément les profils d'intensité de l’ensemble des molécules d'ADN cartographiées, de vérifier la qualité de la cartographie réalisée et, en particulier, de voir où sont situés les segments répliqués au niveau du génome en molécule unique. Nous démontrons la robustesse de notre approche en fournissant, avec une couverture sans précédent (23 311 x), une carte de la réplication de l'ADN bactériophage dupliqué dans des extraits d'œufs Xenopus et mettons en évidence le potentiel du système Irys pour l’étude de la réplication de l'ADN et autres études de génomiques fonctionnelles, en plus de son utilisation standard
DNA replication is a vital process ensuring accurate conveyance of the genetic information to the daughter cells. In eukaryotic organisms, genome replication is carried out by using multiple start sites, also known as replication origins. In metazoans, the mapping of replication remains challenging. Genome wide mapping of human replication origins performed using sequencing techniques only modestly agree. These existing genome wide approaches use large cell populations that smooth out variability between chromosomal copies that could explain this inconsistency. Thus, to get a better understanding of DNA replication and to uncover the cell-to-cell variability, the development of single molecule techniques is fundamental. DNA combing, a widespread technique used to map DNA replication at a single molecule level, is refractory to automation, forestalling genome-wide analysis. To overcome these impediments, we repurposed an optical DNA mapping device based on microfluidics, the Bionano Genomics Irys system, for High-throughput Optical MApping of Replicating DNA (HOMARD). We typically collect, for a single run, over 34 000 images and more than 63 000 Mbp of DNA. Our new open source tools, that required the adaptation of the provided proprietary software, empower us to simultaneously visualize the intensity profiles of all mapped DNA molecules, check the optical mapping performed and, in particular, see where the replication tracks are located genome-wide at a single molecule level. We demonstrate the robustness of our approach by providing an ultra-high coverage (23,311 x) replication map of bacteriophage DNA in Xenopus egg extracts and the potential of the Irys system for DNA replication and other functional genomic studies apart from its standard use
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