Abstract：
The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.
摘要：
基因组包含许多可能经历复杂相互作用并有助于建立的调控元件，维护,和细胞身份的改变。三维基因组组织可以用荧光原位杂交（FISH）在单细胞水平上探索，但是小基因组位点的检测仍然具有挑战性。这里,我们提供了一种快速，简单的方案，用于生成适合检测小基因组元件的明亮FISH探针。我们系统地优化了探针设计和合成，筛选聚合酶的能力，以掺入染料标记的核苷酸，和简化的纯化条件，以产生具有可变阵列（NOVA探针）中染料的纳米镜检查兼容的寡核苷酸。有了这些探测器，我们检测的基因组位点范围从全基因组重复区到千碱基以下的非重复位点。总之,我们引入了一个简单的工作流程来生成密集标记的寡核苷酸池，这有助于检测和纳米级测量单细胞中的小基因组元件。
