一种简捷的Southern印迹杂交方法

A Simple, Rapid Method of Southern Blot Analysis

在综合前人方法的基础上,发展了一种简捷、灵敏、廉价、效果可靠的Southern印迹杂交方案。使用0 4mol LNaOH溶液将琼脂糖凝胶上的DNA在变性的同时转移到尼龙膜上,晾干膜后无需固定就可以直接用于杂交。预杂交液和杂交液选用Church缓冲系统,该缓冲系统中仅需10g LBSA作为封闭剂即可。整个洗膜过程可简化为一种溶液、2～3次洗涤。碱变性及转移后的尼龙膜,也可以用于地高辛(DIG)等非放射性检测系统,并可耐多轮杂交和洗脱。本方法可适用于包括高等植物基因组在内的多种生物材料的DNA检测。

A simple, rapid,sensitive, low-cost and reproducible protocol of Southern blot analysis was developed based on a comprehensive analysis of the previous reports. With a solution of 0\^4 mol/L NaOH, DNA in the agarose gel can be denatured and transferred onto the nylon membrane in a single step. The air-dry membrane can be hybridized directly without fixation. 10 g/L BSA is used as the only blocking reagent in the Church buffer system during the course of pre-hybridization and hybridization. The whole process of washing the membrane can be simplified as one solution, and 2～3 times of washing. The membrane prepared by alkali transferring method can also be used in the non-radioactive DNA detection system such as DIG. Moreover, the blot can be stripped and reprobed for several times. The method described here can be applied to detect target DNA in different species, including higher plants which hold comparatively large genome.