基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

Optimizing and Confirming of Digoxigenin Based Southern Blotting for Wheat Genome Analysis

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 Southern blot analysis based on isotope labeling technique has some shortages such as facility limitation,environmental pollution, and poor stability. Therefore, establishing a safe, stable and efficient Southern hybridization protocol is very important to detect the integration of exogenous genes in transgenic wheat. In this study,by using the DNAs from wild-type wheat and transgenic wheat asmaterial,the DIG-labeled Southern blot anal- ysis was improved through modifying several key steps,including probe preparation and purification, usage of DNA samples amount, digestion system, vacuum transfer conditions, stringent hybridization conditions, and immunoassay detect. The results showed that the purifications of DNA template and probe- labeled could significantly improve the efficiency of the probe labeling when random prime labeling method was used. Desirable digestion result could be a- chieved when 10μg DNA samples with high quality was enzymed in 80μl reaction system for 8 - 12 hours. In the vacuum transfer step,alkaline liquid was better than neutral liquid to obtain clean transmembrane effect. Reagent purity, the temperature and the rotation speed in hybridization inside the hybridization oven impacted on the hybrid- ization results greatly. Application of chemiluminescence detection system combining with improved coating CSPD method was not only easier to operate, but also cleaner in background comparing X-ray imaging optimized. The im- proved digoxigenin-labeled Southern blot technique showed good sensitivity and high ratio of signal to noise forwheat DNA analysis, can be used stably and widely in ordinary laboratory, overcoming restrictions of isotope labeling to experimental conditions, equipments, and physical status of researcher.