Detection of base analogs incorporated during DNA replication by nanopore sequencing
Detection of base analogs incorporated during DNA replication by nanopore sequencing
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DOI：
10.1093/nar/gkaa517
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发表时间：
2020-09-04
影响因子：
14.9
通讯作者：
Egli, Dieter
中科院分区：
文献类型：
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作者：
Georgieva, Daniela;Liu, Qian;Egli, Dieter
DNA合成是细胞增殖和DNA修复的基本要求，但没有一种单一的方法可以高分辨率地识别复制叉的位置、方向和速度。哺乳动物细胞具有在DNA合成期间将胸苷类似物沿着与天然A、T、G和C碱基结合的能力，这允许标记复制或修复的DNA。在这里，我们展示了使用牛津纳米孔技术MinION检测11种不同的胸苷类似物，包括CldU，BrdU，IdU以及单独的EdU或与合成DNA模板中的生物素和其他大体积加合物偶联。我们还表明，大的加合物生物素可以从较小的模拟IdU，这打开了使用类似物的组合，以确定DNA合成的位置和方向的可能性区分。此外，我们检测小鼠多能干细胞基因组中单个DNA分子上的IdU标记，并使用CRISPR/Cas9介导的富集，使用人类线粒体DNA中新合成的DNA链确定复制率。我们的结论是，这种新的方法，被称为微孔测序，有可能在广泛的生物背景下的DNA复制的目标检查。
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 labeling 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 in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.