TAIL-PCR 技术及其在植物基因中克隆.pdfVIP

基因组学与应用生物学，2009 年，第28 卷，第3 期，第544-548 页 Genomics and Applied Biology, 2009, Vol.28, No.3, 544-548 专题介绍 Review TAIL-PCR 技术及其在植物基因中的克隆 1,2 1,2* 2 1,2* 2 郑岑 张立平 唐忠辉 赵昌平 苑少华 1 首都师范大学生命科学学院, 北京, 100048; 2 北京市农林科学院北京杂交小麦工程技术研究中心, 北京, 100097 * 通讯作者, lp-zhang8@; bjhwc2003@ 摘 要 热不对称性PCR (thermal asymmetric interlaced PCR, TAIL-PCR)是一种用来分离与已知序列邻近 的未知DNA 序列的分子生物学技术。该技术利用3 个根据已知序列设计的嵌套特异性引物分别和简并引 物组合进行PCR 反应，选择恰当退火温度对目标片段进行PCR 扩增。TAIL-PCR 技术作为一种使用技术简 单易行，反应高效灵敏，产物特异性高，重复性好，能够在较短的时间内获得目标片段，已经在分子生物学研 究领域广泛应用。本文从TAIL-PCR 技术原理出发，对该技术特异性引物设计、随机引物组合选择、PCR 反 应条件等关键性问题进行综述，并介绍TAIL-PCR 技术在植物基因克隆上的应用现状及发展前景。 关键词 TAIL-PCR, 简并引物, 基因克隆 TAIL-PCR and its Cloning in Plant Gene Zheng Cen 1,2 Zhang Liping 1,2* Tang Zhonghui 2 Zhao Changping 1,2* Yuan Shaohua 2 1 College of Life Science, Capital Normal University, Beijing, 100048; 2 Beijing Engineering Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097 * Corresponding authors, lp-zhang8@; bjhwc2003@ DOI: 10.3969/gab.028.000544 Abstract Thermal asymmetric interlaced PCR (TAIL-PCR) is an efficient technique for isolation of target DNA segments flanking known sequences. Three nested sequence-specific primers and a short arbitrary degenerate (AD) primer were used respectively. Specific products can be amplified through continuous cycles of amplification at different annealing temperature. TAIL-PCR technique is very simple, highly efficient, specificity and sensitivity. The specific PCR products could be successfully amplificated in one day. It has become a powerful tool in a ge- nomic analysis and has been widely used in various fields of molecular biology research. Beginning with the prin- ciple and protocol of TAIL-PCR, methods for special primers design, arbitrary degenerate primers combination, PCR reaction conditions as well as some critical problem are summarized. The applicat