Key Laboratory of Molecular Biophysics of the Ministry of Education, China-UK Joint Lab, School of life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China.
Mol Biol Rep. 2011 Aug;38(6):3629-34. doi: 10.1007/s11033-010-0475-6. Epub 2010 Dec 14.
Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA-enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.
杂交是一种非常重要的分子生物学技术，用于测量 DNA 序列之间遗传相似性的程度，并检测转基因生物中的外源基因。标记 DNA 或 RNA 探针在杂交中起着关键作用。本研究采用碱性磷酸酶非放射性标记 uidA(Gus)DNA 作为探针的方法。在此基础上，我们进一步研究了碱性磷酸酶标记的 uidA(Gus)DNA 探针，用于检测外源 uidA(Gus)基因在转基因黑麦中的转化。对这种 DNA-酶复合物进行了详细的特征和检测，结果表明该探针具有灵敏、特异、安全和经济的特点。通过Southern 印迹杂交和点杂交，在严格条件下以碱性磷酸酶为检测试剂，5-溴-4-氯-3-吲哚磷酸（BCIP）-硝基蓝四唑（NBT）为底物，点杂交表明 uidA(Gus)基因已转化到目标植物中并稳定遗传，Southern 印迹表明至少有两个 uidA(Gus)基因拷贝插入到一条转基因黑麦中。对转基因黑麦的组织化学染色 X-Gluc 也证明了外源 uidA(Gus)DNA 已转化到转基因黑麦中。
