基于荧光染料Cy5.5标记的引物延伸法鉴定细菌RNA剪切加工位点

Fluorescence dye Cy5.5-based primer extension technique determines RNA cleavage sites of bacteria

摘要:

【背景】传统引物延伸法常与放射性标记结合使用，但由于放射性元素半衰期短、危害人体健康及严格的操作要求等限制了其在一般实验室的广泛使用。因此，开发新型非放射性引物延伸法成为当前研究的热点。【目的】建立非放射性的引物延伸法，并利用该方法实现对细菌RNA剪切加工位点的鉴定。【方法】将包含RNA剪切位点的序列克隆至双荧光报告系统，然后利用荧光染料Cy5.5标记的特异性靶向mcherry寡核苷酸引物进行延伸，并辅以Northern blotting、RT-qPCR等技术，确定RNA剪切加工的精确位点。【结果】鉴定了来自解纤维梭菌cip-cel mRNA中的2个剪切加工位点均位于颈环结构下游的单链区域，且在剪切位点附近未发现保守序列。【结论】基于Cy5.5标记的引物延伸法能够精确鉴定RNA剪切加工位点，且与传统的放射性引物延伸法相比，该方法具有更高的安全性和更快的检测速度，能够满足一般实验室的实验要求。

Abstract:

[Background] Traditional primer extension assay usually uses radioactive probes to identify the start sites of transcription and processing sites of transcripts. However, the application of this method in general laboratories is limited because of the short half-life, health hazards, and waste disposal issue of radioisotope-labeled primers. [Objective] To develop a non-radioactive method of primer extension by employing near-infrared fluorescent cyanine dye Cy5.5 and then employ this method to identify the RNA cleavage sites of bacteria. [Methods] The sequence harboring RNA cleavage sites was inserted into the dual-luciferase reporter assay system, and then the Cy5.5-labeled oligonucleotides primer specific to mcherry was used for primer extension. The RNA cleavage sites were precisely identified by Northern blotting and Cy5.5-based primer extension technique. [Results] Two processing sites of cip-cel mRNA were identified, which located in the downstream region of the stem-loop. However, no conserved sequence was identified near the processing sites. [Conclusion] Compared with radioactive isotope-based primer extension methods, the Cy5.5-based primer extension technique not only efficiently determines processing sites of RNA molecules but also avoids the use of hazardous radioactive isotope reagents for labeling and shortens the processing time, which suggests that the Cy5.5-labeled probe is suitable for primer extension assay.