第七章基因操作中的核酸技術(shù)

1、第七章 基因操作中的核酸分析技術(shù)第一節(jié) DNADNA和蛋白質(zhì)相互作用分析一、一、凝膠凝膠阻滯分析（阻滯分析（Gel shift AssayGel shift Assay）, , 該方法用來研究該方法用來研究DNADNA與特異性蛋白的相互作與特異性蛋白的相互作用，通常是放射性標(biāo)記的用，通常是放射性標(biāo)記的DNADNA片段與純化蛋片段與純化蛋白，或提取物中的蛋白混合物相結(jié)合，然白，或提取物中的蛋白混合物相結(jié)合，然后在后在非變性凝膠中非變性凝膠中分析該產(chǎn)物。與游離分析該產(chǎn)物。與游離DNADNA相比，蛋白相比，蛋白-DNA-DNA復(fù)合物的遷移率將降低，復(fù)合物的遷移率將降低，因此，與游離因此，與游離DNA

2、DNA相對應(yīng)，人們將觀察到帶相對應(yīng)，人們將觀察到帶中的中的“阻滯阻滯”。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.) 凝膠阻滯實(shí)驗(yàn)的應(yīng)用鑒定蛋白質(zhì)的提取物中是否存在能同某鑒定蛋白質(zhì)的提取物中是否存在能同某DNADNA結(jié)合的蛋白質(zhì)結(jié)合的蛋白質(zhì)利用利用DNADNA同特定轉(zhuǎn)錄因子的結(jié)合作用通過親同特定轉(zhuǎn)錄因子的結(jié)合作用通過親和層析分離特定轉(zhuǎn)錄因子。和層析分離特定轉(zhuǎn)錄因子。二、Dnase I足跡實(shí)驗(yàn)DNaseDNase I I足跡試驗(yàn)是一種足跡試驗(yàn)是一種鑒別鑒別RNARNA聚合酶等聚合酶等蛋白質(zhì)在蛋白質(zhì)在DN