TurkJBiol 25(2001)405-418 ©TÜB‹TAK Pulsed-FieldGelElectrophoresis(PFGE)Technique anditsuseinMolecularBiology Esin(HACIO⁄LU)BASIM SüleymanDemirelUniversity,FacultyofAgriculture,DepartmentofPlantProtection, 32260Çünür,Isparta-TURKEY HüseyinBASIM AkdenizUniversity,FacultyofAgriculture,DepartmentofPlantProtection, 07058,Antalya-TURKEY Received:30.06.2000 Abstract: Inrecentyears,theuseofpulsed-fieldgelelectrophoresis(PFGE)inthemolecularbiology areahasbeensubjecttomuchresearch.PFGEisapowerfultoolforcharacterizingvariousstrainsat theDNAlevel,obtainingrelevantinformationongenomesizeandconstructingthephysicaland geneticmapofthechromosomeofbacteriathatarepoorlyunderstoodatthegeneticlevelaswellas inseparatingchromosomesinmicroorganisms,andinthelong-rangemappingofmammaliangenes. PFGEalsohasadvantageofexaminingtheelongatedandorientedconfigurationoflargeDNA moleculesinagarosegelsatfinitefieldstrengths.Inthisreview,theuseofPFGEinmolecularbiology, thegeneralcharacteristicsofPFGE,differenttypesofPFGEandfactorsaffectingPFGEare introduced. KeyWords: Pulsed-FieldGelElectrophoresis(PFGE),CHEF,MolecularBiology,Biotechnology, RestrictionEnzymes. Pulsed-FieldJelElektroforez(PFGE)Tekni¤iveMolekülerBiyoloji Alan›ndaKullan›m› Özet: Sony›llarda,molekülerbiyolojialan›ndapulsed-fieldjelelektroforez(PFGE)’inkullan›m›birçok araflt›rmayakonuolmufltur.PFGE,DNAdüzeyindeçeflitlistrainlerinkarakterizeedilmesinde,genom büyüklüklerihakk›ndabilgieldeetmede,genetikdüzeydeanlafl›lamam›flbakterikromozomlar›n›n fizikivegenetikharitalar›n›noluflturulmas›nda,mikroorganizmalar›nkromozomlar›n›nayr›lmas›nda, vememeligenlerininbüyükçaptakiharitalanmas›ndakullan›lanetkilibirmetottur.PFGE;agarozjel içerisindekibüyükDNAmoleküllerininuygunbiralandade¤iflikflekillerdehareketetmesinisa¤layan biravantajadasahiptir.Buderlemede,PFGE’inmolekülerbiyolojialan›ndakullan›m›,PFGE’ingenel özellikleri,çeflitlitiplerivePFGE’ietkileyenfaktörlersunulmufltur. AnahtarSözcükler: Pulsed-FieldJelElektroforez(PFGE),CHEF,MolekülerBiyoloji,Biyoteknoloji, RestriksiyonEnzimler 405 Pulsed-FieldGelElectrophoresis(PFGE)TechniqueanditsuseinMolecularBiology Introduction Muchoftherapidprogressthatisbeingmadeinmolecularbiologytodaydependsuponthe abilitytoseparate,sizeandvisualizeDNAmolecules.Themostcommontechniqueforthis purposeisthatofstandardagarosegelelectrophoresis.Gelelectrophoresis(1)isoneofthe mostcommonlyusedseparationtechniquesinthemodernbiologylaboratory.Itsubiquity arisesfromboththesimplicityandversatilityofthetechnique.Electrophoresishasfound widespreaduseinbiologicalassays,andinthepurificationandseparationofproteinsand nucleicacids.ThephysicalmappingofgenesandDNAsequencingbothdependonseparation bygelelectrophoresis(1).ConventionalgelelectrophoresisofDNAmoleculesiscarriedoutby placingDNAinasolidmatrix(i.e.agaroseorpolyacrylamide)andinducingthemoleculesto migratethroughthegelunderastaticelectricfield.DNAfragmentsfrom100to200basepairs (bp)upto50kilobasepairs(kb)areroutinelyseparatedbyconventionalgelelectrophoresis techniques.Above50kb,becauseofthesizeofthemolecules,thesievingactionofthegelis lost,andfragmentsrunasabroad,unresolvedbandwithanomalouslyhighmobility.Although largerfragments(upto750kb)havebeenresolvedbythistechnique(2),thegelsusedare extremelyfragileduetotheverylowagaroseconcentrations,andtheseparationisnot adequateformostapplications.TheseparationofDNAmoleculesbyothertechniquesistime- consuming.TheneedfortheanalysisoflargeDNAmoleculesisinlarge-scalemapping(3). In1982,Schwartzetal.(4)introducedtheconceptthatDNAmoleculeslargerthan50kb canbeseparatedbyusingtwoalternatingelectricfields(i.e.PFGE).Sincethattime,anumber ofinstrumentsbasedonthisprinciplehavebeendeveloped,andthevalueofusingpulsedfields hasbeendemonstratedforseparatingDNAsfromafewkbtoover10megabasepairs(Mb). ThedevelopmentofPFGEhasincreasedbytwoordersofmagnitudethesizeofDNA moleculesthatcanberoutinelyfractionatedandanalyzed.Thisincreaseisofmajorimportance inmolecularbiologybecauseitsimplifiesmanypreviouslylaboriousinvestigationsandmakes possiblemanynewones.Itsrangeofapplicationspansallorganisms(2)frombacteriaand virusestomammals(5).PFGEhasshownexcellentabilitytoseparatesmall,naturallinear chromosomalDNAsranginginsizefrom50-kbparasitemicrochromosomestomultimillion-bp yeastchromosomes.However,intacthumanchromosomesrangeinsizefrom50millionto 250millionbp(Mb),toolargefordirectPFGEseparations(6).PFGEprovidesthemeansfor theroutineseparationoffragmentsexceeding6,000kb(2,7,8,9).Therefore,PFGE separatesDNAsfromafewkilobase(kb)toover10megabasepairs(Mb)(10). ThenewtechniqueofPFGEtakesadvantageoftheelongatedandorientedconfigurationof largeDNAmoleculesinagarosegelsatfinitefieldstrengths.Animportantbonusofthis techniqueistheeasewithwhichthegenomesizecanbemeasured,aparameterthatwas previouslysubjecttoconsiderableerrorwhenmeasuredbyothertechniques.Oneimportant outcomeoftheuseofPFGEandrestrictionendonucleasedigestionistheconstructionofa physicalmap.GeneralapplicationsofPFGEcanbeintheseparationofwholechromosomes, 406 E.(HACIO⁄LU)BASIM,H. BASIM thelarge-scalerestrictionmappingofchromosomeregionsandinusingDNAfragment purificationasanaidincloning.PFGEwillgreatlyfacilitatethepreciseselectionofverylarge fragmentsforcloning,anditprovidesrapidanalysisofalargechromosomalregion.PFGEhas provenextremelypowerfulintheanalysisoflargeDNAmoleculesfromavarietyofsources, includingspecificallyfragmentedgenomesofbacteria(11),mammals(5),parasiteprotozoa (12-15)andintactchromosomalDNAsfromfungi(16-18).TheintroductionofPFGE techniquesforseparatinglargeDNAmoleculeshashadaninvigoratingeffectonthestudyof chromosomalDNAmolecules,genomestructureandelectrophoreticaltheory. Inthisreview,theuseofPFGEinmolecularbiologythegeneralcharacteristicsofPFGE, differenttypesofPFGEandfactorsaffectingPFGEareintroduced. TypesofPFGE PFGEsizeresolvesDNAmoleculesofalmostamillimeterinlengththroughtheuseof pulsed-fieldelectricfields,whichselectivelymodulatemobilitiesinasize-dependentfashion. Thepulsedelectrophoresiseffecthasbeenutilizedbyavarietyofinstruments(FIGE,TAFE, CHEF,OFAGE,PACEandrotatingelectrodegel)toincreasethesizeresolutionofbothlarge andsmallDNAmolecules(1).ItisimportantwhenchoosingaPFGEsystemtoevaluatecost andperformanceinthelightofprojecteduse.TherearedifferenttypesofPFGE.Theseare: 1.Field-InversionGelElectrophoresis(FIGE):In1986,Carle,FrankandOlsondeveloped asimplersystem,FIGE,inwhichthetwofieldswere180°apart(19).Electrodepolaritywas reversedatintervals,withalongerforwardthanreversepulsetimetogenerateanetforward samplemigration.Netforwardmigrationisachievedbyincreasingtheratioofforwardto reversepulsetimesto3:1.ToimprovetheresolutionofthebandsbyFIGE,thedurationof pulsetimesisincreasedprogressivelyduringarun.Thisiscalled“switchtimeramping”.By changingpulsedurationscontinuallyduringthecourseofanexperiment,FIGEhasthe advantagesofstraightlanesandsimpleequipment.Allthatisneededarestandardgelboxes andapulsecontroller.Today,FIGEisverypopularforsmallerfragmentseparations.FIGE providesacceptableresolutionupto800kilobases(600-750kb). 2.Transverse-AlternatingFieldGelElectrophoresis(TAFE): ThisformofPFGEallows separationoflargeDNAfragmentsinasimple,convenientformatwithoutthedrawbacksof earlierpulsed-fieldtechniques.InTAFE,thegelisorientedverticallyandasimplefour-electrode arrayisplacednotintheplaneofthegel,butinfrontandatthebackofit.Samplemolecules areforcedtozigzagthroughthethicknessofthegel,andalllanesexperiencethesameeffects, sothebandsremainstraight(20).Asthemoleculesmovedownthegel,theyaresubjectedto continualvariationsinfieldstrengthandreorientationangle,buttoalllanesequally.However, theanglebetweentheelectricfieldsvariesfromthetopofthegel(115°)tothebottom (approximately165°)andhencemoleculesstilldonotmoveataconstantvelocityoverthe 407 Pulsed-FieldGelElectrophoresis(PFGE)TechniqueanditsuseinMolecularBiology lengthofthegel.TAFEtechnology,withregularandsharpseparationofDNAbands,willbeof specialadvantageinthestudyofgeneticsofmanypathogenicprotozoans,wheresuchanalysis wasimpossiblebefore(20).TAFEhasbeenusedfortheseparationoffragmentsupto1,600 kilobasefragments. 3.Contour-ClampedHomogeneousElectricFields(CHEF):CHEFisthemostwidelyused apparatus.TheCHEFapparatusprovidesamoresophisticatedsolutiontothedistortingeffects ofboththeedgesofthechamberandthepassiveelectrodes.CHEFhastwenty-fourpoint electrodesequallyspacedaroundthehexagonalcontour.IntheCHEFsystem,thereareno “passive”electrodes.Alltheelectrodesareconnectedtothepowersupplyviaanexternalloop ofresistors,allofwhichhavethesameresistance.Thisloopisresponsibleforsettingthe voltagesofalltheelectrodesaroundthehexagonalcontourtovaluesappropriatetothe generationofuniformfieldsineachofthealternateswitchingpositions.TheCHEFsystemsets thevoltagesatthese24points.Thisapparatusproduceselectricfieldsthataresufficiently uniformsothatalllanesofagelrunstraight.CHEFusesanangleofreorientationof120°with gradiationsofelectropotentialradiatingfromthepositivetothenegativepores.Moleculesup to7,000kbcanbeseparatedbyCHEF(10). 4.Orthogonal-FieldAlternationGelElectrophoresis(OFAGE): Asimilarapparatusthat usedtwononhomogeneouselectricfieldswasreportedbyCarleandOlson(17)in1984.The majordrawbacksoftheseapparatuseswerethatbecausetheelectricfieldswerenotuniform, andtheanglebetweentheelectricfieldvariedacrossthegel,DNAmoleculesmigratedat differentratesdependingontheirlocationinthegel.Thisisespeciallyproblematicin mammaliangenomemapping,whereacontinuousdistributionoffragmentsizesisgenerated. Lane-to-lanecomparisionsandsizeestimationsfordigestedgenomicDNAareless straightforwardwhenfewerdiscretebandsarebeingseparated,aswiththechromosomesof lowerorganismslikeyeast.Theanglebetweentheelectricfieldsvariesfromlessthan180°and