番茄叶片基因组DNA快速制备技术及其在基于实时定量PCR的转基因检测中的应用.pdfVIP

HEREDITAS (Beijing) 2011 9 , 33(9): 1017―1022 ISSN 0253-9772 技术与方法 DOI: 10.3724/SP.J.1005.2011.01017 番茄叶片基因组DNA 快速制备技术及其在基于实时 荧光定量PCR 的转基因检测中的应用 王伟伟, 朱长青, 刘小花, 陈昆松, 徐昌杰 , 310058 以番茄(Solanum lycopersicum L. cv. Micro-Tom) 叶片为试材, 建立了一种简便快速制备叶片基因组DNA 的方法。2~20 mm2 的叶片即可满足制备要求, 制备过程只需一种提取试剂、只涉及1 次移液和1 次离心操作, 不 涉及沉淀。确定了所制备的DNA 用于实时荧光定量PCR 的合适用量为0.1~0.2 L (反应总体积为 12.5 L), 发 现过量模板的使用可降低PCR 效率且可导致扩增失败。该项DNA 快速制备及相适应的实时荧光定量PCR 技 术已成功应用于番茄转基因植株检测。 番茄; DNA 快速制备; 实时荧光定量PCR; 转基因检测 Techniques for rapid preparation of tomato leaf DNA and its appli- cation in real-time quantitative PCR-based transgene detection WANG Wei-Wei, ZHU Chang-Qing, LIU Xiao-Hua, CHEN Kun-Song, XU Chang-Jie Department of Horticulture , College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Abstract: Using tomato (Solanum lycopersicum L. cv. Micro-Tom) leaf as material, a simple and rapid DNA preparation protocol was established. This method required only 220 mm2 leaf with only one extraction solution and involved one pipetation and one centrifugation each. No precipitation was required. The suitable volume of prepared DNA solution, as PCR template, for real-time quantitative PCR was determined to be 0.10.2 L in 12.5 L final reaction volume. The ex- cessive template DNA solution was confirmed to reduce PCR efficiency and even can result in PCR failure. This technique for rapid preparation of DNA and a compatible real-time quantitative PCR were successfully applied in transgene detection of tomato plants. Keywords: tomato; rapid DNA preparation; real-time quantitative PCR; transgene detection 1983 , 20 , , , , 收稿日期: 201012;