Yehuda Yishai, Blumenfeld Britny, Lehmann Dan, Simon Itamar
Dept. of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem.
The Core Research Facility, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem.
J Vis Exp. 2017 Jan 19(119):55157. doi: 10.3791/55157.
Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge. This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.
基因组的复制发生在细胞周期的S期，这是一个高度调控的过程，可确保DNA复制的保真度。每个基因组区域在S期的不同时间通过多个复制起点的同时激活进行复制。复制时间（ToR）与许多基因组和表观遗传特征相关，并与突变率和癌症有关。全面了解健康和疾病状态下复制程序的全基因组情况是未来的一个主要目标和挑战。本文详细描述了“用于绘制基因组复制时间的S/G1拷贝数比”方法（以下简称：CNR-ToR），这是一种绘制哺乳动物细胞全基因组ToR的简单方法。该方法基于S期细胞和G1期细胞之间的拷贝数差异。CNR-ToR方法按6个步骤进行：1. 细胞制备并用碘化丙啶（PI）染色；2. 使用荧光激活细胞分选（FACS）分选G1期和S期细胞；3. DNA纯化；4. 超声处理；5. 文库制备和测序；6. 生物信息学分析。CNR-ToR方法是一种快速简便的方法，可生成详细的复制图谱。
