应用DGGE研究微生物群落时的常见问题分析

462200644ActaMicrobiologicaSinica46(2):3313354April2006:973(G2000026402);(30470054);(50125823)3Tel:[phone];Fax:[phone];E-mail:[e-mail],[e-mail]:(1977-),,,,E2mail:[e-mail]:2005210225;:2005212205;:2005212213DGGE,3(150090):(DGGE),,,DGGE,PCRDGGE,DGGE,DGGE:DGGE;;;;:Q93811:A:000126209(2006)0220331205(Denaturinggradientgelelectrophoresis,DGGE)FischerLerman1979DNA[1]1993Muzyer[2]DGGE,DGGE,,011%1%[3],DGGE,DGGE,,,[4]DGGEDNA,,,,,,PCRDNA:;DNA(RNA);16SrDNAPCR(RT-PCR);(DGGE);;DGGE;;DGGE,,,,DGGE,DGGE,,1DGGEDGGE,DGGEDNA(RNA)PCR(RT2PCR)DGGE,DGGE,111,,DNARNA,,DNA,PCR-DGGEDNADNADNA[5],EDTADNA,(PVPP)(CTAB),CTABDNA,PVPPDNA()(SDS)(K)Krsek[6]++SDSDNADNA,K©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[7]Frosteg¼rd[5]DNADNA,3,,PCR(RT-PCR),,PCR,DNA,,TrisTween220,PCR,[8]112PCRPCR(RT2PCR),PCR,,,DNAPCR,16SrDNA341fP534r(V3)341fP926r(V3V5)968fP1401r(V6V8)Watanabe[9]16SrDNA,DGGE,,16SrDNA,,16SrDNA,,DGGE,200bp(V3),450500bp(V3V5V6V8),,968fP1401341fP926r,DGGEDGGE500bpDNA,50%[10]GC100%[10,11]16SrDNA(),PCR2DGGE,[12],PCR,16SrDNA,PCR(DNA),DNA,PCR,,DNAPCRDNA,Qiu[13]5%PCR,Thompson[14]PCR(ReconditioningPCR)20,10,3,,Zhang[15]PCR3DNA,7molPL(d2PAGE)DNA,DNADGGE,,DNA,(Proofreading)(Highfidelity)113,200bp,8%,500bp6%DGGE500bp,,,200bp,6%12%Tm,Tm,,DNA,,DNAS30%(Tm10),16SV3rDNA30%60%,114DNADGGE,TmDNA5065,WinMeltPMacMelt(http:PP),TGGE2STAR(http:PP)Polandanalysis(http:PP)[16],,,60,,,,Muyzer[1](Timetravelexperiment),DGGE,,Tm233XINGDe-fengetal.PActaMicrobiologicaSinica(2006)46(2)©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(DG2DGGE),,16SrDNA(Multiplemeltingdomains,MMD)DGGE,[17]115DGGE,,SYBRGoldSYBRGreenISYBRGreenII[18],(Ethidiumbromide,EB),,,,EB200,,,,Sanguinetti()Bassam()DGGE,,,,10%15%,[19],,2DGGEDGGE,,,,DGGE,,,,211DGGEDGGE,Gel2ProAnalyzerQuantityOneImageTool,,,,DGGE,DGGE,(Ordination)(Classification),DGGE,(Clustering),(UPGMA)[20](Bio2radGelDocument3000),,,,1(http:PPordination.okstate.eduPoverview.htm),(Multidimensionalscaling,MDS)(Principal2componentanalysis,PCA)DGGE[21](PcoA),,(NMDS),DGGEDGGE,SAS,SSPS,MiniTabStatisticsMDSPCA,,PCA,DGGE,,DGGE,,1Table1OrdinationmethodsClassificationofordinationIndirectgradientDistance2basedEigenanalysis2baseDirectgradientLinearmodelUnimodalmodelPolarordination(PO)(Bray2Curtisordination)Principalcoordinatesanalysis(PcoA)Nonmetricmultidimensionalscaling(NMDS)Principalcomponentsanalysis(PCA)Correspondenceanalysis(CA)Detrendedcorrespondenceanalysis(DCA)Redundancyanalysis(RDA)Canonicalcorrespondenceanalysis(CCA)Detrendedcanonicalcorrespondenceanalysis(DCCA)333:DGGE.P(2006)46(2)©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[22]DGGEDNA,,PCR,d-PAGEPCR,DNA,,Tm,Tm,,,GC,PCRDGGE,,,DGGE,,DGGE[23,24],CHECK-CHIMERA(http:PP35181164152PcgisPchimera.cgi?su=SSU),GenBank,,3DGGEHandelsman[25]1998(,Metagenomics),,[26,27]DGGE,DGGEDNAPCR(Real2timePCR),DGGE,Biolog,,,[28]16SrDNAReal2timePCR16SrRNA,,,DGGE,[1]MuyzerG,SmallaK.Applicationofdenaturinggradientgelelectrophoresis(DGGE)andtemperaturegradientgelelectrophoresis(TGGE)inmicrobialecology.AntonievanLeeuwenhoek,1998,73:127-1411[2]MuyzerG,WaalEC,UitterlindenAG.Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisofpolymerasechainreactionamplifiedgenesencodingfor16SrRNA.ApplEnvironMicrobiol,1993,59:695-7001[3]WardDM,BatesonMM,WellerR,etal.RibosomalRNAanalysisofmicroorganismsastheyoccurinnature.AdvMicrobEcol,1992,12:219-2861[4],,,.DG-DGGE.,2005,25(7):296-3011[5]Frosteg¼rd!,CourtoisS,RamisseV,etal.QuantificationofbiasrelatedtotheextractionofDNAdirectlyfromsoils.ApplEnvironMicrobiol,1999,65:5409-54201[6]KrsekM,WellingtonEMH.ComparisonofdifferentmethodsfortheisolationandpurificationoftotalcommunityDNAfromsoil.JMicrobiolMethods,1999,39:1-161[7]ZhouJ,BrounsMA,TiedjeJM.DNArecoveryfromsoilsofdiversecomposition.ApplEnvironMicrobiol,1996,62:316-3221[8]PurohitHJ,KapleyA,MoharikarAA,etal.AnovelapproachforextractionofPCR-compatibleDNAfromactivatedsludgesamplescollectedfromdifferentbiologicaleffluenttreatmentplants.JMicrobiolMethods,2003,52:315-3231[9]WatanabeK,KodamaY,HarayamaS.DesignandevaluationofPCRprimerstoamplifybacterial16SribosomalDNAfragmentsusedforcommunityfingerprinting.JMicrobiolMethods,2001,44:253-2621[10]MyersRM,FischerSG,LermanLS,etal.NearlyallsinglebasesubstitutionsinDNAfragmentsjoinedtoaGC-clampcanbedetectedbydenaturinggradientgelelectrophoresis.NucleicAcidsRes,1985,13:3131-31451[11]SheffieldVC,CoxDR,MyersRM.Attachmentofa40bpG+Crichsequence(GCclamp)togenomicDNAfragmentsbypolymerasechainreactionresultsinimproveddetectionofsingle-basechanges.ProcNatlAcadSciUSA,1989,86:232-2361[12]ShabirAD,KuenenJG,MuyzerG.NestedPCR-denaturinggradientgelelectrophoresisapproachtodeterminethediversityofsulfate-reducingbacteriaincomplexmicrobialcommunities.ApplEnvir