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

doi:10.3724/SP.J.1005.2011.01017

遗传 ›› 2011, Vol. 33 ›› Issue (9): 1017-1022.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

浙江大学农业与生物技术学院园艺系, 杭州 310058

收稿日期:2010-12-21 修回日期:2011-05-18 出版日期:2011-09-20 发布日期:2011-09-25
通讯作者: 徐昌杰 E-mail:chjxu@zju.edu.cn
基金资助:
国家重点基础研究发展计划(973计划)项目(编号：2011CB100602), 国家自然科学基金重点项目(编号：31030052)和浙江省自然科学基金重点项目(编号：Z3100171)资助

Techniques for rapid preparation of tomato leaf DNA and its application 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

Received:2010-12-21 Revised:2011-05-18 Online:2011-09-20 Published:2011-09-25

摘要/Abstract

摘要： 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 mL (反应总体积为12.5 mL), 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。

关键词: 番茄, DNA快速制备, 实时荧光定量PCR, 转基因检测

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 mL in 12.5 mL 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.

Key words: tomato, rapid DNA preparation, real-time quantitative PCR, transgene detection

王伟伟，朱长青，刘小花，陈昆松，徐昌杰. 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[J]. 遗传, 2011, 33(9): 1017-1022.

WANG Wei-Wei, SHU Chang-Jing, LIU Xiao-Hua, CHEN Hun-Song, XU Chang-Jie. Techniques for rapid preparation of tomato leaf DNA and its application in real-time quantitative PCR-based transgene detection[J]. HEREDITAS, 2011, 33(9): 1017-1022.

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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

Techniques for rapid preparation of tomato leaf DNA and its application in real-time quantitative PCR-based transgene detection

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