高级生物化学与分子生物学专题肿瘤功能基因组学PPT学习教案

1、会计学1高级生物化学与分子生物学专题肿瘤功高级生物化学与分子生物学专题肿瘤功能基因组学能基因组学第1页/共56页第2页/共56页 1 DNA cloning 2 tool enzyme3 target gene4 gene vector第3页/共56页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. Th

2、e 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.第4页/共56页2 tool enzyme restriction endonuclease DNA ligase DNA polymerase I reverse transcriptase polynucleotide k

3、inase end-transferase alkaline phosphatase第5页/共56页structural character of cutting site recognized by restriction enzymerestriction endonucleaserecognized sequence and cutBam H I Eco R I GAATTCCTTAAGGGATCCCCTAGG5Pvu I Sst I GAGCTCCTCGAGCGATCGGCTAGC5Alu I Sma I AGCTTCGACCCGGGGGGCCC5555555555第6页/共56页3

4、target geneThe interested gene is the target gene第7页/共56页 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 librar

5、y. * Polymerase Chain Reaction (PCR).第8页/共56页 synthesize cDNA from mRNAAAAAAA53mRNAAAAAAA35AAAAAATTT.TTT5353primer: oligo dTreverse transcriptaseTTT.TTT53basic hydrolysisTTT.TTT53?TTT.TTT53AAAAAADNA polymerase ITTT.TTT53AAAAAA35S1 nuclease第9页/共56页genomic librarygenomic DNA fragment 50-200kbextractio

6、n restrictively cutgene fragmentsrecombinationrecombinanttransformationgenomic librarytarget genecDNA libraryrecombinationrecombinanttransformationcDNA librarymRNAcDNAdoublestrands第10页/共56页53extension35PCR Process535335denaturationannealing3553533553555335denaturationNext cycle5353第11页/共56页4 gene ve

7、ctor 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. 第12页/共56页 vector* plasmid* cosmid* phage* M13 phage* insect virus DNA (autograph californica virus , ACNPV)* yeast artificial chro

8、mosome DNA* vaccinia virus DNA* simian virus 40 DNA3-10kb40kb29-48.5kb5.243kb 180kb6.407kb20kb4-8kb15kb0.3-1.0kb2.5kb128kb100kb25kb* bovine papilloma virus DNA8.0kb10kb0.2-2.2Mb0.3-1.2Mb* retrovirus DNA* fowlpox virus DNA* adenovirus DNA* herpes simplex virus DNA* cytomegalovirus DNA* Epstein-Barr v

9、irus DNA240kb170kb6.407kb233-238kb8-10kb24-36kb第13页/共56页Xmn I 39662034 Xmn I Pst I 36122067 Pvu II 1424 Ava I 650 Sal I 375 BamH I pBR3224.36kb29 Hind III EcoR I 0A originA screening geneA single restriction siteconditiontetramprori第14页/共56页plasmidpUC192.69kbEco R I Sac IKpn ISma IBam H IXba IHinc I

10、IPst ISph IHind IIIamproripolylinker52bpPlaclac Ilac Z第15页/共56页the procedureof gene cloningseparate targetgene as well as vectorcut target gene and vectorrestrictedlyjoin targetgene and vectorrecombinanttransformationseparate targetgene as well as vectorcut target gene and vectorrestrictedlyligate t

11、argetgene and vectorrecombinantscreeningrecombinantscreeningrecombinanttransformationrecombinantscreeninggo a step further.target geneamplify第16页/共56页incomplete1231+3incomplete digestion123Sma Icomplete1+2+31 - 2, 22 - 5, 33 - 9, 44 - 14, 556n n+n(n+1)/2 n+1 separate target gene第17页/共56页cut and liga

12、te target gene and vectorCGGCCGGCHpa IICCGG GGCCCCGG GGCCHpa IIgenome DNACGGCCGGCHpa IICCGG GGCCCCGG GGCCLigaseCCGGGGCCplasmidHpa II第18页/共56页 recombinant transformation vectors and recombinantscompetent第19页/共56页 recombinant screeningamp or tet etcrestrictionenzememarker-+marker-+第20页/共56页 target gen

13、e amplification第21页/共56页 第22页/共56页 第23页/共56页食管癌细胞食管癌细胞NGALNGAL基因基因55端转录调控区不同长度片端转录调控区不同长度片段段PCRPCR扩增结果扩增结果200bpM 1431 1137 945 657 416 152 11242000bp 1000bp 第24页/共56页重组子重组子pGEM-1431pGEM-1431152152 Xho XhoI I和和BglBglIIII双酶切后，琼双酶切后，琼脂糖凝胶电泳鉴定结果脂糖凝胶电泳鉴定结果 200bpM1 1431 1137 945 657 416 152 1124 M21000bp

14、947 bp 5.0 kb 2.0 kb 第25页/共56页重组子重组子pGLP-1431pGLP-1431152152 Xho XhoI I和和BglBglIIII双酶切后，琼双酶切后，琼脂糖凝胶电泳鉴定结果脂糖凝胶电泳鉴定结果M 1431 1137 945 657 416 1525000bp 1375 bp 564bp 第26页/共56页 target gene expressionprokaryotic expression systemD第27页/共56页Expression analysis of four expression vectors in Expression analy

15、sis of four expression vectors in by by SDS-PAGESDS-PAGE 第28页/共56页eukaryotic expression system21kDa 25kDa1 2 3 4 5 6 7 8 9 10第29页/共56页第30页/共56页第31页/共56页DNA Analysis Southern Blot: telomere length; amplification; deletion PCR: mutation; DNA methylation In situ hybridization: gene location; translocat

16、ionDNA Chip: SNPBioinformatics第32页/共56页DNA Analysis Southern Blot: telomere length; amplification; deletion PCR: mutation; DNA methylation In situ hybridization: gene location; translocationDNA Chip: SNPBioinformatics第33页/共56页 RNA Analysis Northern Blot : expressed genesRT-PCR: expressed genesIn sit

17、u hybridization: gene locationcDNA microarray : differentially expressed genes Bioinformatics第34页/共56页Protein AnalysisWestern Blot: protein concentrationImmunohistochemical Technique: protein location2-D Electrophoresis: differerentially expressed proteinsBioinformatics第35页/共56页Cover of Science19921

18、99319941995199619971998199920002001First MicroarrayPatent Issued1st Catalog GeneChip ProductRoche EasyAccess Commercial launchSacramento2002Roche AmpliChipTM launched2003U133 Set10K19912004100KWorlds First Diagnostic Microarray System Launched by Affy in Europe第36页/共56页11 mMillions of copies of a sp

19、ecificoligonucleotide probeImage of Hybridized GeneChip Array500,000 differentcomplementary probesSingle stranded, labeled targetOligonucleotide probeGeneChip ArrayHybridized Probe Cell1.28cm1.28cm第37页/共56页Perfect Match PMMM Mismatch 独具创新的独具创新的PM-MM探针设计探针设计Oligo 探针探针第38页/共56页ATCGGTAGCCATGCATGAGTTACT

20、AATCGGTAGCCATCCATGAGTTACTA13MisMatch Base第39页/共56页 Discriminate against background signals Increasing sensitivity at low target concentrations第40页/共56页DNARNAGenotyping: Is it A or B?Gene Expression: Which genes? How much?Eukaryotic Cell第41页/共56页One Genechip SystemDNA-GenotypingFamily based linkage s

21、tudies Whole-genome disease associationPharmacogeneticsChromosomal copy numberPopulation geneticsLinkage Disequilibrium ResequencingRNA-Expression AnalysisBiomarker discoveryMechanism of ActionTranscription factor bindingTranscriptome AnalysisDNA methylationAlternative splicing第42页/共56页Whole genome

22、Genotyping Application Genetics analysis-Linkage-Assciation Chromosome copy number analysis-Tumor Amplification & Deletion-other genetics disease第43页/共56页SamplesPolymorphismLow density1-2k SNPsDisequilibriumAssociationLinkageHigh density50k-100k SNPsIn Genes2k-600k SNPsExample: Northern European

23、, Iceland, FinlandClinical Population第44页/共56页TAGCCATCGGTA NSNP 0 PositionC / AGTA C TCAATGATCAGCTATCGGTAGCCAT GATCGGTAGCCAT TATCGGTAGCCAT AATCGGTAGCCAT CCAT G AGTTACTACAT G AGTTACTACAT G AGTTACTACAT G AGTTACTAPM AlleleMM AllelePM AlleleMM AlleleAABBSNP Tiling Strategy第45页/共56页TAGCCATCGGTA NSNP+4 Po

24、sitionC / AGTA C TCAATGATCAGCTGTAGCCAT GGTAGCCAT TGTAGCCAT TGTAGCCAT GCAT G AGTTACTAGTCGCAT C AGTTACTAGTCGCAT G AGTTACTAGTCGCAT C AGTTACTAGTCGPMMMPMMMAAB B+4 Allele+4 Allele+4 Allele+4 Allele第46页/共56页Single Primer Assay Overview250 ng Genomic DNARE digestionXbaXbaXbaAdapter ligationSingle Primer Amp

25、lification4 PCR ReactionsFragmentationand LabelingHyb & Scan on Standard Hardware第47页/共56页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

26、 (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)第48页/共56页全球使用热潮Affyme

27、trix 100K SNP arrays全基因组关联分析多发性硬化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，二型糖尿病全基因组关联分析芬兰东部人群冠心病

28、，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全基因组关联分析糖尿病-Affymetrix, ParAllele and Cambridge Universit