TAIL-PCR的改良及其在分离小麦基因启动子中的应用.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, April 25; 24(4): 695-699 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 技术与方法 TAIL-PCR 1, 2 1 1 1 1 1 仇艳光 , 田景汉 , 葛荣朝 , 赵宝存 , 沈银柱 , 黄占景 1 , 050016 2 , 050081 : 在经典 TAIL-PCR(Thermal asymmetric interlaced PCR) 的基础上, 对其进行了如下四处改进: 用 10 个碱基的 RAPD 引物代替16 个碱基的随机兼并引物作为PCR 中的随机引物; 将较低特异性循环的复性温度由44°C 降至29°C; 增 加 5 个高特异性反应循环, 减少 5 个较低特异性反应循环; 用单引物对第三轮 PCR 产物进行初步鉴定。利用改进的 TAIL-PCR 方法分离了小麦X 基因的 5′未知的侧翼序列, 与GUS 基因融合后转入拟南芥, 通过组织化学检测分析表明 分离到的5′侧翼序列具有启动子功能, 同时说明改进的TAIL-PCR 能更好地应用到较复杂基因启动子的分离。 : 热不对称交错 PCR, 随机引物, 启动子 A Modified TAIL-PCR and Its Application in Isolating Gene Promoter of Wheat 1,2 1 1 1 1 1 Yanguang Qiu , Jinghan Tian , Rongchao Ge , Baocun Zhao , Yinzhu Shen , and Zhanjing Huang 1 College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China 2 Bethuen Military Medical School, Shijiazhuang 050081, China Abstract: Using a modified TAIL-PCR technique, the 5 ′-flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29°C instead of 44°C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles