通过局部变性荧光原位杂交进行基因组寡探针杂交。
Genome oligopaint via local denaturation fluorescence in situ hybridization.
机构信息
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
出版信息
Mol Cell. 2021 Apr 1;81(7):1566-1577.e8. doi: 10.1016/j.molcel.2021.02.011. Epub 2021 Mar 2.
Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.
Cas9 复合物与可编程向导 RNA 靶向特定的双链 DNA 进行切割。通过利用 Cas9 作为基因组 DNA 的可编程超螺旋酶加载器，我们报告了一种称为通过局部变性（GOLD）FISH 的生理温度 DNA 荧光原位杂交（FISH）方法。与传统的 DNA FISH 中的全局变性不同，在 Cas9 产生的切口处加载超螺旋酶允许局部 DNA 变性，减少探针的非特异性结合，并避免诸如热变性等苛刻的处理。GOLD FISH 依赖于 Cas9 切割靶 DNA 序列，并避免了其他依赖于 Cas9 结合的基因组标记方法相关的高核背景。优异的信号亮度和特异性使我们能够对非重复基因组 DNA 基因座进行成像，并分析活性和非活性 X 染色体之间的构象差异。最后，GOLD FISH 可用于快速识别患者组织中的 HER2 基因扩增。
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