基因表达调控-3.pptVIP

* 基因表达与调控 研究基因转录调控的方法 Reporter gene assay EMSA (Electrophoretic mobility shift assay) Foot-pringting Methylation assay Tet/off and Tet/on assay Northern Blotting ChIP SAGE Western blotting assay Chromatin Immunoprecipitation (CHIP) ---- real-time analysis for protein/DNA interaction You must know or have; 1. DNA sequence around interesting bind sites 2. Proteins that could bind to the sites 3. Antibody for the protein Example: RNA Pol II at transcription start point ---Nucleic Acids Research, 2004, 32(11): 1-8 Obtain tissues from animal or human Crosslink chromatin and binding proteins Treat tissues by 1% formaldehyde for 12 min and then stop reaction by 0.125M Glycine Disaggregate tissue by homogenizer, collect cells Break cells by buffer containing 0.5% NP-40, spin to collect nuclei Break nuclei by buffer containing 1% SDS Sonicate chromatin into ~500bp fragments Block protein A/G agarose by lDNA, tRNA and BSA Incubate sonicated DNA with blocked proteinA/G agarose to remove non-specific bind, spin to get supernatant, save sample as “Input” Incubate pre-cleaned supernatant with anti-RNA Pol II antibody Add blocked protein A/G agarose into reaction Spin to collect protein A/G agarose and wash Elute chromatin DNA by 1%SDS, 100mM NaHSO3 Incubate to reverse crosslink and use the sample as PCR temperate Design PCR primers that flack the binding site (200~500bp) 15. Rea-time PCR Serial Analysis of Gene Expression (SAGE) ----分析特定细胞或组织中的基因表达谱 SAGE is based on the following principle: 9 bp DNA tag contains sufficient information to uniquely identify a mRNA NNNN 44=256 NNNNN 45=1024 NNNNNN 46=4096 NNNNNNN 47=16376 NNNNNNNN 48=65504 NNNNNNNNN