变性梯度凝胶电泳-转载.ppt

2011年9月17日变性梯度凝胶电泳(denaturedgradientgelelectrophoresis，DGGE)最初是Fisher和Lerman等人于20世纪80年代初期发明的，起初主要用来检测DNA片段中的点突变。Muyzer等人在1993年首次将其应用于微生物群落结构研究。后来又发展出其衍生技术，温度梯度凝胶电泳（temperaturegradientgelelectrophoresis，TGGE）。此后十年间，该技术被广泛用于微生物分子生态学研究的各个领域，目前已经发展成为研究微生物群落结构的主要分子生物学方法之一。变性梯度凝胶电泳（DGGE）:一种分离相似大小DNA片段的电泳方法。即双链DNA在变性剂(如尿素和甲酰胺)浓度或温度梯度增高的凝胶中电泳，随变性剂浓度升高，由于Tm值不同，DNA的某些区域解链，降低其电泳泳动性，导致迁移率下降，从而达到分离不同片段的目的。由于各类微生物（如细菌和古细菌）的16sRNA基因序列中可变区的碱基顺序有很大的差异，其中不同土壤微生物的16sRNA基因的V3区扩增的DNA片断在DGGE中的应用最为广泛，根据电泳条带的多寡和条带的位置可以初步辨别出样品中微生物的种类多少，粗略分析土壤样品中微生物的多样性。Themethodsofdetectingsinglebasemutations1.Southernblotting2.Single-StrandConformationalPolymorphism(SSCP,单链构象多态性分析)3.DenaturingGradientGelElectrophoresis(DGGE,变性凝胶梯度电泳)4.Carbodiimide(CDI，碳化二亚胺检测法)5.ChemicalCleavageofMismatch(CCM，化学切割错配法)6.Rnasecleavage（RNA酶裂解法）7.Heteroduplexanalysis（异源双链分析）8.theProteinTruncationTest(PTT，蛋白截短测试)9.TemporalTemperatureGradientGelElectrophoresis(TTGE,时间温度梯度电泳).yearpercent%number20014.75%2120023.85%1720036.79%3020049.05%40200511.09%49200612.90%57200715.84%70200815.16%67200913.12%5820107.47%33inall100%4420.00%2.00%4.00%6.00%8.00%10.00%12.00%14.00%16.00%18.00%出版年2001200220032004200520062007200820092010①thepaperswhichtitlescontain‘DGGE’in2001-2010ThearticleaboutDGGEyearPercent%number20014.63%18420024.48%17820036.06%24120047.52%29920059.23%367200610.99%437200713.13%522200815.39%612200918.86%75020109.73%387inall100%39770.00%2.00%4.00%6.00%8.00%10.00%12.00%14.00%16.00%18.00%20.00%②thepaperswhichthemescontain‘DGGE’in2001-20102008-20101.MicrobiologicalcharacterisationofRobioladiRoccaveranocheeseusingPCR-DGGE.Bonetta,S.Carraro,E.Rantsiou,K.Cocolin,L.2008FoodMicrobiology.(IF2.847)2.Variationintheactivediazotrophiccommunityinricepaddy-nifHPCR-DGGEanalysisofrhizosphereandbulksoil.Wartiainen,I.;Eriksson,T.;Zheng,W.W.;Rasmussen,U.2008AppliedSoilEcology.(IF2.247)3.Analysisofcommunitystructureofamicrobialconsortiumcapableofdegradingbenzo(a)pyrenebyDGGE.Luo,Y.R.;Tian,Y.;Huang,X.;Yan,C.L.;Hong,H.S.;Lin,G.H.;Zheng,T.L.2009MarinePollutionBulletin(IF2.63)4.BulksoilandrhizospherebacterialcommunityPCR-DGGEprofilesandbeta-galactosidaseactivityasindicatorsofbiologicalqualityinsoilscontaminatedbyheavymetalsandcultivatedwithSilenevulgaris(Moench)Garcke.Martinez-Inigo,M.J.;PerezSanz,A.;Ortiz,I.;Alonso,J.;Alarcon,R.;Garcia,P.;Lobo,M.C.Chemosphere(IF3.253)20095.Applicationofreal-timePCR,DGGEfingerprinting,andculture-basedmethodtoevaluatetheeffectivenessofintrinsicbioremediationonthecontrolofpetroleum-hydrocarbonplume.Kao,C.M.;Chen,C.S.;Tsa,F.Y.;Yang,K.H.;Chien,C.C.;Liang,S.H.;Yang,C.A.;Chen,S.C.2010JournalofHazardousMaterials(IF4.144)6.Comparativeanalysesofampliconmigrationbehaviorindifferingdenaturinggradientgelelectrophoresis(DGGE)systems.Thornhill,D.J.;Kemp,D.W.;Sampayo,E.M.;Schmidt,G.W.2010CoralReefs(IF3.056)7.Characterizationandbiotechnologicalpotentialofpetroleum-degradingbacteriaisolatedfromoil-contaminatedsoils.Zhang,Z.Z.;Gai,L.X.;Hou,Z.W.;Yang,C.Y.;Ma,C.Q.;Wang,Z.G.;Sun,B.P.;He,X.F.;Tang,H.Z.;Xu,P.BioresourceTechnology(IF4.253)Inadenaturinggradientacrylamidegel,double-strandedDNAissubjectedtoanincreasingdenaturantenvironmentandwillmeltindiscretesegmentscalledmeltingdomains.Themeltingtemperature(Tm)ofthesedomainsissequence-specific.WhentheTmofthelowestmeltingdomainisreached,theDNAwillbecomepartiallymelted,creatingbranchedmolecules.PartialmeltingoftheDNAreducesitsmobilityinapolyacrylamidegel.InDGGE,thedenaturingenvironmentiscreatedbyacombinationofuniformtemperature,typicallybetween50and65℃andalineardenaturantgradientformedwithureaandformamide.Asolutionof100%chemicaldenaturantconsistsof7Mureaand40%formamide.SincetheTmofaparticularmeltingdomainissequence-specific,thepresenceofamutationwillalterthemeltingprofileofthatDNAwhencomparedtowild-type.DNAcontainingmutationswillencountermobilityshiftsatdifferentpositionsinthegelthanthewild-type.Ifthefragmentcompletelydenatures,thenmigrationagainbecomesafunctionofsize(Fig1)Fig.1.AnexampleofDNAmeltingpropertiesinaperpendiculardenaturinggradientgel.Atalowconcentrationofdenaturant,theDNAfragmentremainsdouble-stranded,butastheconcentrationofdenaturantincreases,theDNAfragmentbeginstomelt.Then,atveryhighconcentrationsofdenaturant,theDNAfragmentcancompletelymelt,creatingtwosinglestrands.Thedenaturinggradientmaybeformedperpendicularorparalleltothedirectionofelectrophoresis.（1）Aperpendiculargradientgel,inwhichthegradientisperpendiculartotheelectricfield,typicallyusesabroaddenaturinggradientrange,suchas0–100%or20–70%.（2）InparallelDGGE,thedenaturinggradientisparalleltotheelectricfield,andtherangeofdenaturantisnarrowedtoallowbetterseparationoffragments.ExamplesofperpendicularandparalleldenaturinggradientgelswithhomoduplexandheteroduplexfragmentsareshowninFigure2Fig.2.A.PerpendiculardenaturinggradientgelB.ParalleldenaturinggradientgelThemeltingbehaviourofDNAfragments,aswellastheoptimalgradientcanbedeterminedexperimentallywithperpendiculargradientgels.Perpendiculargelshaveanincreasinggradientofdenaturantsortemperaturefromlefttoright,perpendiculartothe