Genome-Wide Single-Molecule Analysis of Replication Kinetics

 描述（由申请人提供）：DNA复制的时间和空间模式是基因组生物学的基本方面。它们与转录调控、染色质修饰、核结构和基因组进化的模式相关。此外，随着细胞分化，复制时间发生变化，复制时间的中断与基因组的不稳定性相关，这表明复制时间和基因组代谢的其他重要方面之间存在密切关系。然而，目前用于测定复制动力学的技术的局限性限制了该领域的进展。绘制复制动力学的基因组方法存在分辨率低和灵敏度低的问题，妨碍了对单个复制起点的鉴定。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，全基因组复制映射技术，我们称之为光学复制映射。这种方法将结合联合收割机体内标记复制的DNA与最先进的兆碱基大小的单个DNA分子的光学映射，使我们能够可视化DNA复制模式的数万个单独的染色体片段覆盖基因组的30倍的深度。光学复制技术的成功发展将使我们和其他研究小组能够回答有关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人员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。

 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.