常用的实验技术-张华屏.pptVIP

常用的实验技术 科研实验中心 张华屏 DNA水平的研究 遗传性疾病 限制性片段多态性(RFLP) 表观遗传学 DNA甲基化 组蛋白修饰 （染色质免疫共沉淀） 点多态性实验方法 1.DNA的提取 2.PCR 3.限制性内切酶酶解 4.聚丙烯酰胺凝胶电泳分离 5.染色 Step 1: Crosslinking stabilizes protein-DNA complexes Step 2: Cell Lysis removing cytosolic protein can help reduce background and increase sensitivity Step 3: Chromatin Preparation DNA fragmentation Step 4: Immunoprecipitationenriches for the protein-DNA complex of interest Step 5: Crosslinking Reversal and DNA Clean-up the crosslinks between protein and DNA are reversed Step 6: DNA Quantitation Step 1: Crosslinking Step 2: Cell Lysis The lysis stage extracts the crosslinked protein-DNA complexes from cells or tissue and brings them into solution. At this stage, cellular components are liberated by dissolving the cell membrane with detergent based solutions. Because protein-DNA interactions occur primarily in the nuclear compartment, removing cytosolic protein can help reduce background and increase sensitivity. The presence of detergents or salts will not affect the protein-DNA complex, as the covalent crosslinking achieved in step one will keep the complex stable throughout the ChIP procedure. Step 3: Chromatin Preparation The extraction step yields all nuclear material, which includes unbound nuclear protein, full length chromatin and the crosslinked protein-DNA complexes. In order to analyze protein binding sequences, the extracted genomic DNA must be sheared into smaller, workable pieces. DNA fragmentation is usually achieved either mechanically by sonication or enzymatically by digestion with micrococcal nuclease (MNase). Step 4: Immunoprecipitation To isolate a specific modified histone, transcription factor or co-factor of interest, ChIP validated antibodies are used to immunoprecipitate and isolate the target from other nuclear components. This step selectively enriches for the protein-DNA complex of interest and eliminates all other unrelated cellular material