原位
荧光原位杂交
基因组
染色质
计算生物学
DNA
生物
杂交探针
DNA测序
自体荧光
原位杂交
生物物理学
荧光
化学
遗传学
物理
光学
基因
染色体
有机化学
基因表达
作者
Yanxiang Ni,Bo Cao,Tszshan Ma,Gang Niu,Yingdong Huo,Jian‐Dong Huang,Danni Chen,Yi Liu,Bin Yu,Michael Q. Zhang,Hanben Niu
出处
期刊:eLife
[eLife Sciences Publications Ltd]
日期:2017-05-09
卷期号:6
被引量:35
摘要
High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.
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