第七章基因操作中的核酸技术讲述.pptx

第七章 基因操作中的核酸分析技术 第一节 DNA和蛋白质相互作用分析 一、凝胶阻滞分析（Gel shift Assay）, 该方法用来研究DNA与特异性蛋白的相互作用，通常是放射性标记的DNA片段与纯化蛋白，或提取物中的蛋白混合物相结合，然后在非变性凝胶中分析该产物。与游离DNA相比，蛋白-DNA复合物的迁移率将降低，因此，与游离DNA相对应，人们将观察到带中的“阻滞”。 Figure 7-29. A gel-mobility shift assay. The principle of the assay is shown schematically in (A). In this example an extract of an antibody-producing cell line is mixed with a radioactive DNA fragment containing about 160 nucleotides of a regulatory DNA sequence from a gene encoding the light chain of the antibody made by the cell line. The effect of the proteins in the extract on the mobility of the DNA fragment is analyzed by polyacrylamide-gel electrophoresis followed by autoradiography. The free DNA fragments migrate rapidly to the bottom of the gel, while those fragments bound to proteins are retarded; the finding of six retarded bands suggests that the extract contains six different sequence-specific DNA-binding proteins (indicated as C1–C6) that bind to this DNA sequence. (For simplicity, any DNA fragments with more than one protein bound have been omitted from the figure.) In (B) the extract was fractionated by a standard chromatographic technique (top), and each fraction was mixed with the radioactive DNA fragment, applied to one lane of a polyacrylamide gel, and analyzed as in (A). (B, modified from C. Scheidereit, A. Heguy, and R.G. Roeder, Cell 51:783–793, 1987.) 凝胶阻滞实验的应用 鉴定蛋白质的提取物中是否存在能同某DNA结合的蛋白质 利用DNA同特定转录因子的结合作用通过亲和层析分离特定转录因子。 二、Dnase I足迹实验 DNase I足迹试验是一种鉴别RNA聚合酶等蛋白质在DNA上结合位点的方法，它不仅能找到与特异性DNA结合的目标蛋白，而且能告知目标蛋白结合在哪些碱基部位。  三、体内足迹实验 DMS 硫酸二甲酯 四、酵母单杂交 lacZ 五、染色质免疫共沉淀