[自然科学]techniqueofmolecularbiology

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1、MolecularBiologyoftheGene,6/E---Watsonetal.(2007)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods1TherevisedcentraldogmaTranslationRNAprocessingDNArepairandrecombination基因组的保持基因组的表达2Chapter20T

2、echniquesofMolecularBiology3TechniquesofMolecularBiologyseparatingindividualmacromolecularsfromthemyriadmixturesincelldissectingthegenomeintomanageablesizedsegmentsformanipulationandanalysisofspecificDNAsequencesTechniquesofmolecularbiologyhavebeenoneofthema

3、jordrivingforcesintheresearchofmolecularbiology4Themethodsdependuponunderstandingofthepropertiesofbiologicalmacromoleculesthemselves.Hybridization(分子杂交）---thebase-pairingcharacteristicsofDNAandRNADNAcloning---DNApolymerase,restrictionendonucleasesandDNALigas

4、ePCR---ThermophilicDNApolymerase5OutlineTopic1:NucleicacidsTechniquesTopic2:ProteinTechniquesTopic3:TechniquesforstudyingDNA-proteininteractionTopic4:Techniquesforstudyingprotein-proteininteractionBasicprincipleBasicprocedureapplication6Topic1:NucleicacidsTe

5、chniquesSeparationbyElectrophoresis(电泳分离)CutbyRestrictionendonuclease(限制性内切酶切割)IdentificationbyHybridization(杂交鉴定)DNACloningandgeneexpression(基因克隆和表达技术）PCR（聚合酶链式反应）7GelelectrophoresisseparatesDNAandRNAmoleculesaccordingtosize,shapeandtopologicalproperties1.G

6、elElectrophoresis（凝胶电泳）81.DNAandRNAmoleculesarenegativelycharged,thusmoveinthegelmatrix(胶支持物)towardthepositivepole(正电极)2.LinearDNAmoleculesareseparatedaccordingtosizes.ThelargeDNAmoleculesmoveslowerthanthesmallmolecules.DNAgelmobility(electrophoreticmobility

7、，DNA在胶上的迁移率)9largemoderatesmallRungel(跑胶)103.ThemobilityofcircularDNAmoleculesisaffectedbytheirtopologicalstructures.ThemobilityofthesamemolecularweightDNAmoleculewithdifferentshapesis:supercoiled(超螺旋)>linear(线性)>nickedorrelaxed(缺刻或松散)11DNAcanbevisualizedbys

8、tainingthegelwithfluorescentdyes,suchasethidiumbromide(EB,溴化乙锭)DNAisseparatedbygelelectrophoresis1kb0.5kb2kb3kb4kbDNAmarker12Gelmatrix(胶支持物)isajello(果冻状）-likeporousmaterialthatsupportsandallowsm