State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of Chemistry and Chemical Engineering, Hunan University , Changsha 410082, P. R. China.
Anal Chem. 2017 Mar 21;89(6):3445-3451. doi: 10.1021/acs.analchem.6b04312. Epub 2017 Mar 6.
Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics. We develop a novel fluorescence in situ hybridization (FISH) strategy based on a new branched hybridization chain reaction (bHCR) for efficient signal amplification in the FISH assay and a ligase-mediated discrimination for specific mutation detection. To our knowledge, this is the first time that HCR has been realized for mutation detection in the FISH assay. In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA, and it generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR. Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA. Moreover, this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications.
超灵敏和特异的基因表达的原位成像对于分子医学和临床治疗诊断至关重要。我们开发了一种基于新的分支杂交链式反应（bHCR）的新型荧光原位杂交（FISH）策略，用于在 FISH 检测中进行有效的信号放大，并通过连接酶介导的区分用于特定突变检测。据我们所知，这是首次在 FISH 检测中实现了 HCR 用于突变检测。体外实验表明，连接-bHCR 策略在区分 mRNA 中的单核苷酸变异方面具有很高的特异性，并且它产生了高度分支的聚合产物，比 HCR 具有更高的扩增效率或更好的灵敏度。成像分析表明，连接-bHCR 为单个 mRNA 分子的定位产生了高度明亮的点状信号，并且不同颜色的点状信号在单个 mRNA 的点突变基因分型中具有高度特异性。此外，该策略显示出在单细胞水平上定量成像 mRNA 表达的潜力。因此，该策略可能为开发用于各种诊断和研究应用的高灵敏度和特异的 FISH 方法提供了一个新的有前途的范例。
