第七章基因操作中的核酸技术

1、第七章 基因操作中的核酸分析技术第一节 DNADNA和蛋白质相互作用分析一、一、凝胶凝胶阻滞分析（阻滞分析（Gel shift AssayGel shift Assay）, , 该方法用来研究该方法用来研究DNADNA与特异性蛋白的相互作与特异性蛋白的相互作用，通常是放射性标记的用，通常是放射性标记的DNADNA片段与纯化蛋片段与纯化蛋白，或提取物中的蛋白混合物相结合，然白，或提取物中的蛋白混合物相结合，然后在后在非变性凝胶中非变性凝胶中分析该产物。与游离分析该产物。与游离DNADNA相比，蛋白相比，蛋白-DNA-DNA复合物的迁移率将降低，复合物的迁移率将降低，因此，与游离因此，与游离DNA

2、DNA相对应，人们将观察到带相对应，人们将观察到带中的中的“阻滞阻滞”。Figure 7-29. A gel-mobility shift assay. The principle of the assay is shown schematically in (A). In this example an 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-produ

3、cing cell line is mixed with a radioactive DNA fragment containing about 160 nucleotides of 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 t

4、he cell line. The effect of the 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 proteins in the extract on th

5、e 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 by autoradiography. The free DNA fragments migrate rapidly to the bottom of the gel, while tho

6、se fragments bound to proteins are retarded; the finding of six retarded bands suggests that the extract contains six different sequence-proteins are retarded; the finding of six retarded bands suggests that the extract contains six different sequence-specific DNA-binding proteins (indicated as C1sp

7、ecific DNA-binding proteins (indicated as C1C6) that bind to this DNA sequence. (For simplicity, any DNA fragments 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 stan

8、dard with more than one protein bound have been omitted from the figure.) In (B) the extract was fractionated by a standard chromatographic technique chromatographic technique (top),(top), and each fraction was mixed with the radioactive DNA fragment, applied to one lane of a and each fraction was m

9、ixed 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, polyacrylamide gel, and analyzed as in (A). (B, modified from C. Scheidereit, A. Heguy, and R.G. Roeder, CellCell 51:783 51:783793, 793, 1987.) 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鉴定蛋白质的提取物中是否存在能同某DNADNA结合的蛋白质结合的蛋白质利用利用DNADNA同特定转录因子的结合作用通过亲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。和层析分离特定转录因子。二、Dnase I足迹实验DNaseDNase I I足迹试验是一种足迹试验是一种鉴别鉴别RNARNA聚合酶等聚合酶等蛋白质在蛋白质在DN