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1、APPLIEDANDENVIRONMENTALMICROBIOLOGY,Jan.2001,p.190±197Vol.67,No.10099-2240/01/$04.0010DOI:10.1128/AEM.67.1.190±197.2001PhylogeneticSpeci®cityandReproducibilityandNewMethodforAnalysisofTerminalRestrictionFragmentPro®lesof16SrRNAGenesfromBacterialCommunities121JOHNDUNBAR,*L
2、AWRENCEO.TICKNOR,ANDCHERYLR.KUSKE12BiosciencesDivisionandTechnologyandSafetyAssessmentDivision,LosAlamosNationalLaboratory,LosAlamos,NewMexico87545Received25July2000/Accepted10October2000Terminalrestrictionfragment(TRF)analysisof16SrRNAgenesisanincreasinglypopularmethodfo
3、rrapidcomparisonofmicrobialcommunities,butanalysisofthedataisstillinadevelopmentalstage.WeassessedthephylogeneticresolutionandreproducibilityofTRFpro®lesinordertoevaluatethelimitationsofthemethod,andwedevelopedanessentialanalysistechniquetoimprovetheinterpretationofTRFdat
4、a.ThetheoreticalphylogeneticresolutionofTRFpro®leswasdeterminedbasedonthespeci®cityofTRFspredictedfrom3,90816SrRNAgenesequences.WithsequencesfromtheProteobacteriaorgram-positivedivision,asmuchas73%oftheTRFswerephylogeneticallyspeci®c(representingstrainsfromatmosttwogenera
5、).However,thefractiondecreasedwhensequencesfromthetwodivisionswerecombined.ThedatashowthatphylogeneticinferencewillbemosteffectiveifTRFpro®lesrepresentonlyasinglebacterialdivisionorsmallergroup.TheanalyticalprecisionoftheTRFmethodwasassessedbycomparingninereplicatepro®les
6、ofasinglesoilDNAsample.Despitemeticulouscareinproducingthereplicates,numeroussmall,irreproduciblepeakswereobserved.Asmanyas85%ofthe169distinctTRFsfoundamongthepro®leswereirreproducible(i.e.,notpresentinallninereplicates).Substantialvariationalsooccurredintheheightofsynony
7、mouspeaks.Tomakecomparisonsofmicrobialcommunitiesmorereliable,wedevelopedananalyticalprocedurethatreducesvariationandextractsareproduciblesubsetofdatafromreplicateTRFpro®les.TheprocedurecanalsobeusedwithotherDNA®ngerprintingtechniquesformicrobialcommunitiesormicrobialgeno
8、mes.ComparativeanalysisofcomplexmicrobialcommunitiesinTheTRFmethodhasbeenusedsuccessfullyfordiff
