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《Acta Agriculturae Shanghai》 2001-02
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CLONING OF FOREIGN GENE'S FLANKING SEQUENCES IN TRANSGENIC RICE BY INVERSE PCR

Han Zhiyong Wang Xinqi Shen Gezhi (Crop Breeding and Cultivation Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106)  
On the basis of inverse PCR, we have founded a IPCR technique to clone foreign gene's flanking sequence in transgenic rice, which is suitable to treat mass materials. We used mini preparation protocol to extract total DNA of transgenic rice, and DNA was digested by more than 10 times restriction endonuclease overnight in 50μL. The digested fragments were purified and self ligated in a reaction volume of 20 μL. In order to enhance the specificity of PCR amplifying flanking sequences, the nested PCR was used, and hot start PCR and touchdown PCR were combined with it. Thirty five of foreign gene's flanking sequences have been cloned in transgenic rice, the cloned fragments, sizes are 300~750 bp, and the specificity of PCR product has been proved by Southern blot. The result showed that this IPCR technique is quick, convenient and stable for flanking sequence cloning.
【Fund】: 国家“8 6 3”计划!(10 1- 0 4- 0 1- 0 6 );; 上海市农业科学院青年科技基金项目!(2 0 0 0 - 0 1)
【CateGory Index】: S511
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