基于 Cy5.5 标记的 DNA 探针的荧光核酸斑点杂交方法的建立。

Development of fluorescence-based nucleic acid blot hybridization method using Cy5.5 labeled DNA probes.

机构信息

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, Shanxi Province, China.

Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, Shanxi Province, China.

出版信息

J Microbiol Methods. 2022 Jun;197:106479. doi: 10.1016/j.mimet.2022.106479. Epub 2022 Apr 30.

DOI:10.1016/j.mimet.2022.106479

PMID:35504367

Abstract

Near-infrared (NIR) fluorophores are widely used as fluorescent probes for bioimaging because of their minimal photodamage to biological samples, deep penetration, and low interference from background autofluorescence. Here, we employed a NIR fluorescent cyanine dye Cy5.5 to label DNA probes for nucleic acid blot hybridization. The specificity and sensitivity of fluorescent DNA probes were proven by both Southern blot and Northern blot using cellulolytic bacterium Ruminiclostridium cellulolyticum as a model. Furthermore, employing the method, we successfully identified the gene disruption of ClosTron to rule out off-target, analyzed the differential transcription of genes under different conditions, and confirmed RNA cleavage. Compared to other nonradioactive probes, the preparation and detection of Cy5.5-labeled probes are more simple, more economical, and versatile, suggesting that the Cy5.5-labeled probes are suitable for nucleic acid blot hybridization in addition to bioimaging.

摘要

近红外（NIR）荧光团由于对生物样品的光损伤最小、穿透深度深以及背景自发荧光的干扰低，因此被广泛用作荧光探针用于生物成像。在这里，我们使用近红外荧光花菁染料 Cy5.5 标记 DNA 探针用于核酸印迹杂交。通过使用纤维素分解菌 Ruminiclostridium cellulolyticum 作为模型的 Southern blot 和 Northern blot，证明了荧光 DNA 探针的特异性和灵敏度。此外，采用该方法，我们成功地鉴定了 ClosTron 的基因缺失以排除脱靶，分析了不同条件下基因的差异转录，并证实了 RNA 切割。与其他非放射性探针相比，Cy5.5 标记探针的制备和检测更简单、更经济、更通用，表明 Cy5.5 标记探针除了可用于生物成像外，还适用于核酸印迹杂交。