启动子活性分析课件

1、启动子活性分析基因表达调控的研究策略和常用方法 2015-9-22 基因表达调控是多层次的复杂过程启动子（Promoters） 是位于结构基因5端上游的DNA序列，其长度从100 bp到200 bp不等，与转录起始时RNA聚合酶识别、结合和启动转录有关。启动子控制基因转录的起始时间和表达的程度。启动子的组成promoter普遍转录因子启动子活性分析 Reporter Assay EMSA ChIP（一）氯霉素乙酰转移酶基因（CAT）可催化乙酰CoA的乙酰基转移到氯霉素3羟基，而使氯霉素解毒。线性范围较窄，灵敏性较低 （二）半乳糖苷酶 由大肠杆菌lacZ基因编码，可催化半乳糖苷水解。 （三） 荧

2、光素酶（Luciferase） 是自然界中能够产生生物荧光的酶的统称。荧光的产生是来自于荧光素（底物）的氧化，哺乳细胞无内源性荧光素酶。 最常用的荧光素酶有细菌荧光素酶、萤火虫荧光素酶和Renilla荧光素酶。萤火虫荧光素酶灵敏度高，检测线性范围宽，是最常用于哺乳细胞的报告基因。（四）荧光蛋白家族：GFP, BFP,RFP promoterluc缺失（deletion）突变（mutation）荧光素酶报告基因检测Gel Shift AssayElectrophoretical Mobility Shift AssayEMSABand Shift Assay1. Introduction2. P

3、rinciple3. Application4. Materials5. Procedure6. Proof of SpecificityContentsDNA-protein complexes migrate slower than non-bound DNA in a native polyacrylamide or agarose gel, resulting in a “shift” in migration of the labeled DNA band.PrincipleR. Voll 09/01A double-stranded oligonucleotide containi

4、g a NF-B- binding site is labeled with a radioactive isotope and incubated with different nuclear extract.During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift compared to the free probe.NF-BFree ProbeRadioactively labeled oligonucleotidewith NF-B - binding site (probe) and bo

5、und NF-B complexesRadioactively labeled oligonucleotide with NF-B - binding site (probe)Nuclear extract of non-activated cellsNuclear extract ofactivated cellsApplicationDetection of DNA-binding factors/proteins1. Analysis of DNA sequences (e. g. promoter or enhancer regions) for their potential to

6、bind specifically to proteins/nuclear extracts 2. Analysis of (sub-)cellular extracts for the presence of certain DNA-binding proteins (e. g. a transcription factor with a known recognition sequence)Materials3 or 5 end-labeled DNA target.Protein extractunlabeled DNA targetPolyacrylamide gel in 0.5X

7、TBEElectrophoresis apparatusPositively charged nylon membraneElectroblotter transfer apparatusUV lampX-ray filmPrepare the ProbeR. Voll 09/01AP-1biotin-5-GCT TGA TGA CTC AGC CGG AA C-3 3-CGA ACT ACT GAG TCG GCC TT G-5-biotinNF-kBbiotin-5-AGT TGA GGG GAC TTT CCC AGG C-3 3-TCA ACT CCC CTC AAA GGG TCC

8、G-5-biotin Binding motif1. Synthesize complementary single stranded biotin-labeled and unlabeled oligonucleotides containing a transcription factor binding site.2. Annealing the Oligos：Heat up an equimolar mixture of the 2 oligos to 95C and let them slowly cool down by turning off the heat block.Pla

9、n Binding ReactionsR. Voll 09/01 Procedure1. Prepare nuclear protein extracts2. Prepare Biotin- labeled and unlabeled target DNA3. Plan Binding Reactions4. Prepare and Pre-Run Gel5. Prepare and Perform Binding Reactions6. Electrophorese Binding Reactions7. Electrophoretic Transfer of Binding Reactio

10、ns to Nylon Membrane8. Cross-link Transferred DNA to Membrane9. Detect Biotin-labeled DNA by ChemiluminescenceProof of SpecificityR. Voll 09/011. Supershift using antibodies against the DNA-binding protein ( protein Specificity )2. Competition for binding to the labeled probe using unlabeled wildtyp

11、e and mutated oligos ( DNA Specificity ) R. Voll 09/01Competition with Unlabeled OligosIncreasing amounts of unlabeled oligos containing the NF-kB binding siteor unlabeled oligos with a mutated binding site were added to the reaction mixprior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.Free probep50/p65Wild type oligo Mutated oligoGGG GAC TTT CCCGGA GAC TTT CCCChromatin immunoprecipitation Used to determine whether a given protein binds to a given DNA sequence in vivoLike all protein analysis involving anti