应用RT-PCR检测基因的体外转录活性

A Method Based on RTPCR for Analysis of theTranscriptional Activity of Gene in vitro

体外转录分析已广泛应用于分子生物学领域．由于体外转录所产生的ＲＮＡ的量极少，需有灵敏的方法检测生成的ＲＮＡ．本研究发展了一种基于ＲＴ－ＰＣＲ技术鉴定体外转录产物的方法．将待研究基因的启动区与任何一段已知的含转录起始位点的编码序列相连，制备成体外转录的模板．体外转录后，用ＤＮａｓｅⅠ将ＤＮＡ模板完全降解；生成的ＲＮＡ经反转录合成ｃＤＮＡ；再以ｃＤＮＡ为模板，用相应的引物进行ＰＣＲ扩增，产物用琼脂糖凝胶电泳检测．该法灵敏度高，操作简便，无需同位素，且体外转录模板制备方便．还可结合其他技术，如Ｓｏｕｔｈｅｒｎ印迹分析，能获得更高的灵敏度．

For a more sensitive method to determine the RNA product,a method based upon RTPCR was developed to detect the RNA product generated from in vitro transcription.To prepare the template for in vitro transcription,the promoter region was ligated with a known sequence containing transcriptional initial site.After transcription,the DNA template was completely degraded by DNaseⅠ(RNasefree)and the generated RNA was reversally 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.In this procedure,it is unnecessary to prepare the probe and the template is easier to prepare.Moreover,this method can be combined with other procedures,such as Southern blot,to increase the sensitivity and give quantitative data.