卤虫体外转录模型及其转录活性检测

通过改进的细胞核提取方法,获取了纯度较高的细胞核并建立了卤虫体外转录系统模型。将待基因与含转录起始位点的报告基因的编码序列相连,制备成体外转录模块。由于体外转录所产生的RNA量极少,需要一种灵敏的方法检测生成的RNA。应用了RT－PCR技术鉴定体外转录产物的方法能有效解决这一问题。转录后,用无RNase的DNaseI将DNA模板完全降解;生成的RNA经逆转录反应合成cDNA,再以cDNA为模板,用相应的引物进行PCR扩增,琼脂糖凝胶电泳检测结果。该法灵敏度高,操作简单安全,无需同位素。

Artemia in vitro Transcription System and the Detection of Transcriptional Activity

Analysis of in vitro transcription has been widely used in microbiological research. Through the developed method, high purity Artemia nuclei is isolated and Artemia in vitro transcriptional system is founded. To prepare the template for in vitro transcription, the promoter region was ligated with a known sequence containing transcriptional initial site. Since the quantity of the generated RNA was low, we developed a sensitive method basing on the RT PCR to detect the RNA product. After transcription, the DNA template was completely degraded by DNaseI (RNase free) and the generated RNA was revers transcribed to cDNA. The synthesized cDNA was then amplified by PCR and, finally the product was analyzed by agarose gel electrophoresis. The advantages of this method are sensitive, simple, and it can avoid using radioisotope.