口腔疾病分子生物学

MolecularBiologyofOralDiseasesSpeaker:WangLizhenCollegeofStomatologySchoolofMedicineShanghaiJiaoTongUniversityContentsandObjectivesFundamentsofmolecularbiology1Majorexperimentaltechniquesofmolecularbiology23Applicationsoffundamentsandtechniquesofmolecularbiologyintheresearchingoforalpathogens45SummaryBiologicalbasisoforalgeneticdisordersPartIFundamentsofMolecularBiologyWhatismolecularbiology?Whatgenesaremadeof?Howdogeneswork?Question1:Whatismolecularbiology?DefinitionThefieldofbiologywhichstudiesthestructureandfunctionofgenesatamolecularlevel.MethodsGeneticsBiologicalchemistryQuestion2:Whatgenesaremadeof?DNAMajorityRNAMinorityMid-19thCenturyMendel’sLawsofInheritance(孟德尔的遗传法则)GregorMendel,1822-18841869Nuclein(核素)DeoxyribonucleicAcid-DNA(脱氧核糖核酸)ProteinRibonucleicAcid-RNA(核糖核酸)FriedrichMiescher,1844-18951910TheChromosomeTheory(染色体学说)ThomasHuntMorgan,1866-1945ChromosomeGenesDNAProteinRNA???1944GenesOswaldAvery,1877-1955DNAQuestion3:Howdogeneswork?HowGenesAreReplicated(复制)?HowGenesDirecttheProductionofRNAsandPolypeptides(多肽)?HowGenesAccumulateMutations(突变)?Work1:HowGenesAreReplicated?DNA--Doublehelix(双螺旋)JamesWatsonandFrancisCrick,1953FourBases(碱基)Adenine(A),Guanine(G),Cytosine(C),Thymine(T)Work1:HowGenesAreReplicated?SemiconservativeReplication(半保留复制)A+T,G+CWork1:HowGenesAreReplicated?Work2:HowDoGenesproduceRNAs&Polypeptides?GeneExpression(基因表达)Transcription(转录)Translation(翻译)GeneExpressionTranscriptionDNARNApolymerase(聚合酶)mRNATranslationmRNARibosomes(核糖体)Polypeptides(Protein)Work3:HowDoGenesAccumulateMutations?TypesSmall-scalemutationsPointmutations–DNAreplication–Transitions–transversionsInsertionsDeletionsLarge-scalemutationsinchromosomalstructureChromosomaltranslocationsInterstitialdeletionsChromosomalinversionsLossofheterozygosityWork3:HowDoGenesAccumulateMutations?ImplicationMutationEvolution(进化)BiodiversityDisordersCentralDogmaNewHotTopic:Epigenetics(表观遗传学)HumanEpigenomeProject(HEP)(2003)(人类表观基因组计划)X-chromosomeDosageCompensation(X染色体剂量补偿)DNAMethylation(甲基化)HistoneModifications(组蛋白修饰)•SamegenesbutadifferentdinkinthetailFunctionalAnnotationoftheMammaliangenomeFANTOMProject(2000)“Thegreatestintellectualrevolutionofthelast40yearsmayhavetakenplaceinbiology.Cananyonebeconsiderededucatedtodaywhodoesnotunderstandalittleaboutmolecularbiology?”--F.H.WestheimerPartIIMajorTechniquesofMolecularBiologyGeneCloning(基因克隆)NucleicAcidHybridization(核酸杂交)PolymeraseChainReaction(PCR,聚合酶链反应)Technique1:GeneCloning(基因克隆)DefinitionProcedureConditionsToolEnzymesRestrictionEndonucleaseDNALigaseVectorsLeadinTransformationTransfectionTransductionHostsDefinitionWhatisgene?-TheunitofhereditythatismadeofaDNAsequenceoccupyingaspecificlocationonachromosomeandcodesforapolypeptidechain.Whatisclone?-Apopulationofidenticalmolecules(genes),cellsororganisms,allofwhicharederivedfromthesameparentbyasexualmeans.Whatisgenecloning?-Theprocessofproducinggeneticallysimilarmolecules,cells,ororganismsfromacommonprecursorbyasexualreproductioninvitroorinvivo.BaseSequences(碱基顺序)FunctionsMutations~1973InsulinGeneDefinitionInsulinGeneBacterialHosts(细菌宿主)RecombinantDNA(重组DNA)InsulinGenePlasmids/PhageDNAs(Vectors)(质粒/噬菌体)(载体)GeneCloningInsulinProteinDefinition1973~1973StanleyCohen&HerbertBoyerThefirstcloningexperiment1980JohnGurdonClonesofidenticalfrogs1997IanWilmutAsheepnamedDollyTechnique1:GeneCloning(基因克隆)DefinitionProcedureConditionsToolEnzymesRestrictionEndonucleaseDNALigaseVectorsLeadinTransformationTransfectionTransductionHostsProcedureConstructingaGeneLibrary(建立基因文库)IdentifyingtheDesiredClone(分离目的基因)AnalysingtheDesiredGene(分析目的基因)Step1:ConstructingaGeneLibraryGeneLibraryDefinition-AcollectionoflivingbacterialcoloniesthathavebeentransformedwithdifferentpiecesofDNAfromtheorganismthatisthesourceofthegeneofinterest.TypesGenomic(基因组)DNALibrarycDNALibraryGenomicDNALibrarypurifyinggenomicDNArestrictionnuclease(限制性内切酶)addingtoplasmidvectorgenomicDNAclonegenomicDNAlibraryCellularGenomicDNAStep2:IdentifytheDesiredCloneProbes(探针)Polynucleotides/ligonucleotides(多核苷酸)/(寡核苷酸)Antibodies(抗体)Step3:AnalysetheDesiredGeneThegeneThegene’sproductsTechnique1:GeneCloning(基因克隆)DefinitionProcedureConditionsToolEnzymesRestrictionEndonucleaseDNALigaseVectorsLeadinTransformationTransfectionTransductionHostsCondition1:ToolEnzymesRestrictionEndonucleases(限制性内切酶)DNALigase(连接酶)Enzyme1:RestrictionEndonucleasesOriginE.Coli(大肠杆菌)StewartLinn&WernerArber,1960sRolesForeign(外源性)DNA:CutUpHostCell’sOwnDNA:Restrict•Advantages1.Whereverthissequenceoccurs,andonlywhenthissequenceoccurs,theenzymewillmakeacut.Table1RecognitionSequencesandCuttingSitesofSelectedRestrictionEndonucleasesAluIAG↓CTBamHIG↓GATCCEcoRIG↓AATTCHindIIGTPy↓PuACHindIIIA↓AGCTTHpaIIC↓CGGKpnIGGTAC↓CPstICTGCA↓GSalIG↓TCGACSmaICCC↓GGGXmaIC↓CCGGGNotIGC↓GGCCGCEnzyme1:RestrictionEndonucleases2.Stickyends(黏性末端)Bluntends(平齐末端)•AdvantagesEnzyme1:RestrictionEndonucleasesEnzyme2:DNALigaseDNALigaseCovalentBonds(共价键)Fun