Genome-Wide Single-Molecule Analysis of Replication Kinetics

复制动力学的全基因组单分子分析

• 批准号: 8969943
• 负责人: NICHOLAS R RHIND
• 金额: $ 25.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-16 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8969943
• 关键词: Address; B-Cell Development; Benchmarking; Biological Assay; Biology; Cell physiology; Cells; Chromosome Structures; Chromosomes; DNA; DNA Replication Timing; DNA biosynthesis; Data; Development; Erythropoiesis; Evolution; Fiber; Fire - disasters; Flow Cytometry; Future; Genetic Transcription; Genome; Genomic Instability; Genomic approach; Genomics; Globin; Goals; Heavy-Chain Immunoglobulins; Hematopoietic; Heterogeneity; Human; Human Genome; IGH@ gene cluster; Individual; Kinetics; Label; Length; Location; Maps; Metabolism; Mus; Nature; Noise; Nuclear Structure; Optics; Pattern; Physiologic pulse; Regulation; Replication Initiation; Replication Origin; Research; Resolution; S Phase; S-Phase Fraction; Saccharomycetales; Signal Transduction; Site; Staging; Stem cells; Structure; Surveys; Techniques; Technology; Thymidine; Time; Transcriptional Regulation; analog; cell type; cellular development; chromatin modification; genome analysis; genome-wide; hydroxyurea; improved; in vivo; mathematical model; prevent; programs; public health relevance; repaired; research study; single molecule; technology development; tool

 DESCRIPTION (provided by applicant): The temporal and spatial patterns of DNA replication are fundamental aspects of genome biology. They correlate with patterns of transcriptional regulation, chromatin modification, nuclear structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of genome metabolism. However, the limitations of current techniques for assaying replication kinetics are limiting progress in the field. Genomic approaches to mapping replication kinetics suffer from low resolution and low sensitivity, preventing the identification f individual replication origins. Single locus techniques are laborious, restricting the number of experiments than can feasibly be done. We propose to develop an efficient, high-throughput, single-molecule, genome-wide replication mapping technology that we call optical replication mapping. This approach will combine in vivo labeling of replicated DNA with state-of-the-art optical mapping of megabase-sized single DNA molecules, allowing us to visualize patterns of DNA replication on tens of thousands of individual chromosomal fragments covering the genome to a thirty-fold depth. Successful development of optical replication technology will allow us and other groups to answer fundamental questions about DNA replication kinetics. Furthermore, it will provide accurate information about replication timing for workers in many other fields, essential to understand how replication timing influences other critical aspects of genome metabolism.

 描述(申请人提供)：DNA复制的时间和空间模式是基因组生物学的基本方面。它们与转录调节、染色质修饰、核结构和基因组进化的模式相关。此外，随着细胞的分化，复制时间会发生变化，而复制时间的中断与基因组的不稳定性相关，这表明复制时间与基因组代谢的其他重要方面之间存在密切关系。然而，目前用于分析复制动力学的技术的局限性限制了该领域的进展。基因组方法用于绘制复制动力学图存在分辨率低和灵敏度低的问题，阻碍了对单个复制起始点的识别。单基因座技术是费力的，限制了实验的数量，而不是可行的。我们建议开发一种高效、高通量、单分子、全基因组的复制映射技术，我们称之为光学复制映射。这种方法将复制DNA的体内标记与最先进的兆库大小的单DNA分子的光学映射相结合，使我们能够可视化覆盖基因组的数万个单独染色体片段上的DNA复制模式，深度达到30倍。光学复制技术的成功开发将使我们和其他小组能够回答有关DNA复制动力学的基本问题。此外，它将为许多其他领域的工作者提供有关复制时机的准确信息，这对于了解复制时机如何影响基因组代谢的其他关键方面至关重要。