Detection of Base Analogs Incorporated During DNA Replication by Nanopore Sequencing

DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labelling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU, as well as, EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules, incorporated during DNA replication in the genome of mouse pluripotent stem cells. We conclude that this novel method has the potential for genome-wide examination of DNA replication in stem cell differentiation or cell transformation.

DNA合成是细胞增殖与DNA修复的核心必要前提，但目前尚无单一方法能够以高分辨率识别复制叉的位置、行进方向与移动速度。哺乳动物细胞在DNA合成过程中，可将胸苷类似物（thymidine analogs）与天然的A、T、G、C碱基一同掺入DNA链，这一特性可用于标记复制或修复中的DNA。本研究利用牛津纳米孔科技（Oxford Nanopore Technologies）MinION测序仪，在合成DNA模板中检测到11种不同的胸苷类似物，涵盖CldU、BrdU、IdU以及单独存在的EdU，或与生物素（Biotin）及其他大体积加合物偶联的EdU。我们还证实，可将大体积加合物生物素与小分子类似物IdU区分开来，这为使用类似物组合来确定DNA合成的位置与方向提供了可能。此外，我们在小鼠多能干细胞的基因组中，检测到了DNA复制阶段掺入单条DNA分子的IdU标记。综上，该新型方法有望实现干细胞分化或细胞转化过程中全基因组范围内的DNA复制分析。