博士研究生课高级生物化学与分子生物学专题肿瘤功能基因组学许丽艳.ppt

Progress of Functional Genomics,Li-Yan Xu,DNA Recombination Technology,Gene Chip,Strategy of Functional Genomics,RNA interference,DNA recombination technology,1 DNA cloning 2 tool enzyme 3 target gene 4 gene vector,1 DNA cloning It is a process of DNA molecular amplification. Usually, the first a target DNA fragment is inserted to a vector and a recombinant (replicon) is constructed. The second the recombinant is transformed into host cell and screen out the cell containing the recombinant. The last that cell is amplified, namely a mass of target DNA molecule is gained.,2 tool enzyme restriction endonuclease DNA ligase DNA polymerase I reverse transcriptase polynucleotide kinase end-transferase alkaline phosphatase,3 target gene The interested gene is the target gene,source of the target gene * It is from genomic DNA directly, this is prokaryotic gene only generally. * It is from artificial synthesis, this is simple polypeptide gene generally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).,cDNA library,transformation,cDNA library,extension,4 gene vector The gene vectors are DNA molecules, which structure is reconstructed. They can carry target DNA fragment The target gene or DNA fragment is amplified and expressed.,vector,separate target gene,cut and ligate target gene and vector,target gene expression,prokaryotic expression system,D,Expression analysis of four expression vectors in E.coli by SDS-PAGE,eukaryotic expression system,DNA Recombination Technology,Gene Chip,Strategy of Functional Genomics,RNA interference,DNA Analysis Southern Blot: telomere length; amplification; deletion PCR: mutation; DNA methylation In situ hybridization: gene location; translocation DNA Chip: SNP Bioinformatics,DNA Analysis Southern Blot: telomere length; amplification; deletion PCR: mutation; DNA methylation In situ hybridization: gene location; translocation DNA Chip: SNP Bioinformatics,RNA Analysis Northern Blot : expressed genes RT-PCR: expressed genes In situ hybridization: gene location cDNA microarray : differentially expressed genes Bioinformatics,Protein Analysis Western Blot: protein concentration Immunohistochemical Technique: protein location 2-D Electrophoresis: differerentially expressed proteins Bioinformatics,Discriminate against background signals Increasing sensitivity at low target concentrations,Whole genome Genotyping Application,Genetics analysis -Linkage -Assciation Chromosome copy number analysis -Tumor Amplification & Deletion -other genetics disease,TAGCCATCGGTA N,SNP 0 Position,C / A,GTA C TCAATGATCAGCT,SNP Tiling Strategy,Single Primer Assay Overview,250 ng Genomic DNA,RE digestion,Xba,Xba,Xba,Screening and testing of SNPs for association with BMI. Genome-wide SNPs (86,604) were screened using parental genotypes to find those likely to affect offspring BMI. The top 10 SNPs from the screening step (ranked by power from most likely to least likely) are shown. These SNPs were tested using offspring genotypes for association with BMI using the FBAT. The rs7566605 SNP is highlighted in bold.,Screening and testing of SNPs for association with BMI (SCIENCE VOL 312 14 APRIL 2006),全球使用热潮Affymetrix 100K SNP arrays,全基因组关联分析多发性硬化 Multiple Sclerosis -Serono Ltd. Identifies 80 Genes Involved in Multiple Sclerosis Using 100,000 SNPs -Comprising a total of 900 people with MS and 900 healthy individuals in French, Swedish and American populations 全基因组关联分析芬兰东部人群冠心病，二型糖尿病coronary heart disease and type 2 diabetes -Jurilab Ltd. made ground-breaking discoveries in coronary heart disease and type 2 diabetes -allow Jurilab to develop revolutionary predisposition tests.“ 全基因组关联分析高血压，急性心肌梗塞 hypertension&Acute Myocardial Infarction -Jurilab found more than 300 new hypertension genes discovered. -Jurilab discovers more than 200 new genes in Acute Myocardial Infarction. 全基因组关联分析糖尿病 -Affymetrix, ParAllele and Cambridge University Collaborate For Large-Scale Genetic Study of Diabetes -1,000 control samples and 1,000 diabetic samples,GeneChip Expression Array Design,Gene Sequence,Multiple oligo probes,Perfect Match,Mismatch,5,3,Tissue(Cell) 1 Tissue (Cell) 2,Total RNA Total RNA,PolyA+mRNA PolyA+mRNA,Probe 1 Probe 2,Microarray 1 Microarray 2,Analysis,Identification,ChIP: Mapping of DNA Binding Factors,Assay uses chromatin IP to generate labeled DNA fragments Assay success depends on antibody quality Chromatin-protein Immunoprecipitation Purify; amplify and label DNA fragments Data analysis may be easier than typical transcription assay Use non-specific IP control for comparison Signal regions typically more well defined,Target,RefSeq,OMIM,SWALL (Swissprot/treMB