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

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0.1~0.2μL(反应总体积为12.5μL),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。

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

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 μL in 12.5 μL final reaction volume. The excessive 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.