第三章+3-2+目的基因的获得.ppt

* remove excess probe and autoradiograph Differential filter hybridization reverse transcribe radiolabeled cDNA transfer to two nylon filters hybridize filters with radiolabeled cDNA * Filter hybridization (continued) localize differentially expressed cDNA clones on plate compare films and pinpoint unique hybridization signals identify by DNA isolation and sequencing * * Probe I Probe II AAAAA TTTTT 5’ plate phage on lawn of E. coli putative clone of differentially expressed RNA two filter copies Probe I: cDNA from infected tissue cDNA synthesis Probe II: cDNA from healthy tissue 1 2 library construction 3 4 Differential hybridization * AAAAA TTTTT 5’ cDNA synthesis 1 2 library construction purify protein X derive antibody lacZ AAA cDNA Probe plate phage on lawn of E. coli cDNA clone for protein X 3 filter copy, expose protein 5 4 7 6 Screening with antibodies Lambda expression vector * AAAAA TTTTT 5’ cDNA synthesis 1 2 library construction Purify protein X Derive partial amino acid sequence X AAA cDNA Label and Probe plate phage on lawn of E. coli cDNA clone for protein X 3 filter copy 5 4 7 6 Reverse translation Lambda vector Reverse translation Trp-Met-His-Lys-Ser-Gly TGCNNNBLABLANNNCTGAA * Differential gene expression developmentally regulated gene expression stimulus-induced gene expression tissue- or cell type-specific gene expression complexity of the tissue! * Analysis of differentially expressed genes identification quantification differential filter hybridization differential display PCR (DD-PCR) cDNA subtraction cDNA RDA SSH Northern blotting reverse Northern blotting RNase protection cDNA (micro)array hybridization * Northern blotting Probe labeling pBS-SemaIII dATP dGTP dTTP dCTP RNA isolation AAAAAA Gel electophoresis Blotting Hybridization Autoradiography 第三章 目的基因的获得－目的基因的分离克隆及其方法 * 第三章 目的基因的获得 二.根据mRNA的特异性分离目的基因 （一）mRNA differential display PCR (DDRT PCR) * 差式显示的基本策略是通过反转录与PCR扩增mRNA中特定的一小部分，用DNA序列分析胶（6％－8％）同步分离显示扩增产物以进行比较。