论文部分内容阅读
以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。
The transgenic wheat and wild-type wheat DNA were used to optimize the Southern hybridization analysis of wheat genomic DNA with digoxigenin, including probe preparation and purification, amount of DNA in the sample, enzyme digestion system, India conditions, hybridization conditions, immune detection methods. The results showed that the labeling efficiency of the template was significantly improved by the random primer-labeled template and the labeled probe. 10μg of high-quality DNA sample was obtained after 8-12h in 80μl system. When the membrane was changed, the alkaline membrane was cleaner than that of the neutral membrane. The purity of the reagent, the temperature of the hybridization and the speed of the hybridization oven all had a significant influence on the hybridization performance. With the improved CSPD coating method, the chemiluminescence detection system Compared with the simple X-ray imaging, it is easier to operate and the background is cleaner. The Southern hybridization analysis of digoxigenin-labeled wheat in this study shows high sensitivity and signal-to-noise ratio with stable results and can overcome the influence of isotope labeling on experimental conditions , Equipment and laboratory personnel such as physical limitations, in general laboratory application.