第四章分子生物学研究法修定版(13年)

1、51069. 1)10670%301ln()99. 01ln(N动画动画*ACTTCGACAA1:ACTTCG2:AACTTCGACTTCGAACTTCGACAACTTCGACAA 3:ACACTACTTACTTCACTTCGAC4:ACACTTCACTTCGAC现在多使用不同颜色的现在多使用不同颜色的荧光剂标记双脱氧核苷荧光剂标记双脱氧核苷酸，然后于一个体系内酸，然后于一个体系内反应并一起电泳，通过反应并一起电泳，通过颜色不同读取顺序颜色不同读取顺序凝胶滞缓实验凝胶滞缓实验动画动画cDNA差示分析法（书差示分析法（书)不确切：接头的加法与短接不确切：接头的加法与短接头的去除头的去除Yeast

2、 two-hybrid system:a genetic assay for detecting protein-protein interactionsXYSuppose we have two proteins.One way of answering this question is touse the yeast two hybrid method.To understand the method we must first consider how gene expression is regulated in yeast.and the question,do these two

3、protein bind each other?Regulation of gene expression in yeastgeneUAS(upstream activation sequence)transcription activatorDNA binding domainactivation domaintranscription machineryand now back to the question.Yeast two-hybrid system:a genetic assay for detecting protein-protein interactionsXYDo thes

4、etwo proteinbind?Regulation of gene expression in yeastgeneUAS(upstream activation sequence)transcription activatorDNA binding domainactivation domaintranscription machineryreporter geneWe start with yeast possessing a reporter gene (a genemaking a product that is easy to detect).And now we introduc

5、e genes coding for two hybridproteins.Yeast two-hybrid system:a genetic assay for detecting protein-protein interactionsHis, -galXYRegulation of gene expression in yeastgeneUAS(upstream activation sequence)transcription activatorDNA binding domainactivation domaintranscription machineryreporter gene

6、XYXYXYdo we get expression of the reporter?yes, we have expression ofthe reporter indicating thatthe proteins bind!Do theseproteinsbind?our two hybridproteins bind!thus forming a functionaltranscription activator.Yeast two-hybrid system:a genetic assay for detecting protein-protein interactionsHis,

7、-gal后基因组研究焦点：基因功能研究反义寡核苷酸(antisense oligodeoxynucleotides)技术,是指利用人工合成的反义RNA和反义DNA来阻断基因的转录或复制 抑制基因表达抑制基因表达观察功能变化观察功能变化KnockOut 法法Antisense反义寡核苷酸法法Ribozyme法法指有催化活性的指有催化活性的RNA RNAi法法3.2.1RNAi 发现历程：1990年，曾有科学家给矮牵牛花插入一种催生红色素的基因，希望能够让花朵更鲜艳。但意想不到意想不到的事发生了：矮牵牛花完全褪色，花瓣变成了白色！? ?1995年 康奈尔大学 Guo等在对线虫C.elegans的研

8、究中，应用正义链RNA作为对照，研究par-1基因的表达，意意外的发现外的发现，正义链RNA与反义链RNA同样能够抑制目的基因的表达。 3.2.2RNAi机制 Dicer酶酶断裂dsRNA产生小干涉RNA risc):一种一种RNA-蛋白质复合物蛋白质复合物,通过与目标通过与目标mRNA完全或者部分的互补配对来实施切割或者翻译抑制功能完全或者部分的互补配对来实施切割或者翻译抑制功能Sirna解螺旋解螺旋 与靶与靶mRNA结合结合 靶靶mRNA断裂断裂Sense-正义链正义链antisense 反义链反义链short-interfering RNAQuickTime and aGIF decompressorare needed to see this picture.加工长链加工长链 dsRNA形成形成 21-23 nt 小片段小片段Tuschl, 2001siRNAslong dsRNA