DNA聚合酶

MINI-REVIEW

OverviewofthermostableDNApolymerasesforclassicalPCRapplications:frommolecularandbiochemicalfundamentalstocommercialsystems

KayTerpe

Received:22July2013/Revised:20September2013/Accepted:22September2013/Publishedonline:1November2013#Springer-VerlagBerlinHeidelberg2013

AbstractDuringthegenomicsera,theuseofthermostableDNApolymerasesincreasedgreatly.Manywereidentifiedanddescribed—mainlyofthegeneraThermus,ThermococcusandPyrococcus.Eachpolymerasehasdifferentfeatures,resultingfromoriginandgeneticmodification.However,therationalchoiceoftheadequatepolymerasedependsontheapplicationitself.ThisreviewgivesanoverviewofthemostcommonlyusedDNApolymerasesusedforPCRapplication:KOD,Pab(Isis™),Pfu,Pst(DeepVent™),Pwo,Taq,Tbr,Tca,Tfi,Tfl,Tfu,Tgo,Tli(Vent™),Tma(UITma™),Tne,Tthandothers.KeywordsThermostableDNApolymerase.Polymerasechainreaction(PCR).Extensionrate.Errorrate.Half-lifetime.Extensiontemperature

Introduction

ItisamatteroffactthatthermostableDNApolymeraseswith5′→3′amplificationactivityareoneofthekeyenzymesinmanymolecularapplications.Therefore,industrialbusinessvolumeismuchandthereisfinancialpressuretofindbetterpolymerasesthantheestablishedones.MainlyA-typeandB-typepolymerasesareinuse(Table1).Theincreasingmicrobi-ologygenomeprojectsincombinationwithnewmoleculartechnologiesfacilitatelaunchingnewthermostableDNApoly-merases.However,marketingnameslikesuper,turbo,ultra,dream,goldandmanymoreconfusetheusersandarenothelpful.Publishedvaluesoffidelityaremostlyanalysedatoptimalconditionswithλ-DNA.Theseerrorratescannottrans-mitforanyapplication.APCRprocessmustoftenbevalidated

K.Terpe(*)

SensoQuestGmbH,Hannah-Vogt-Str.1,37085Göttingen,Germanye-mail:k.terpe@sensoquest.deURL:www.sensoquest.de

beforebecomingstandard.Timeandtemperatureofdenaturingcandeactivatethepolymerasemoreorlessdependingontheusedenzyme.FactorslikeDNAorigin,primerandproductlengthaswellasguanine–cytosinecontentshouldhaveadirectinfluenceonthechoiceofpolymerase(Wuetal.1991).Salt,magnesiumanddeoxyribonucleotidetriphosphate(dNTP)con-centrationscangreatlyaffectthePCR(Lingetal.1991;Owczarzyetal.2008).AdditiveslikeBSA(Al-SoudandRådström2001),dimethylsulfoxide(ChesterandMarshak1993),formamide(Sarkaretal.1990),betaine(Henkeetal.1997;Reesetal.1993),ethyleneglycoland1,2-propanediol(Zhangetal.2009),andothers(Al-SoudandRådström2000;VaradarajandSkinner1994)areinusetooptimizespecificityandPCRamplification.Mostoftheseimportantfactorsarerecommendedforcalculationsofthetheoreticalannealingtem-perature.Basisofthiscalculationisthemeltingtemperature.Manyformulasareusedaddictedinsimilarbutnotidenticalresults(Table2).Therealoptimalannealingtemperatureisveryoftendeterminedbyusingathermalcyclerwithtemperaturegradient.Additional,theheatingandcoolingratesofthethermalcycler,thethermalconductivityofthethermoblockmaterial,thevolumeofthemixture(ChangandLee2005)andthethicknessoftheusedplasticmaterialhaveanimportantinfluenceontheevaluationofanoptimalPCRprocess(Table3).Theperfor-manceofthethermalcyclersaswellasthetransferofprotocolsfromonesystemtoanotherarealsoimportantpointswhichcouldbeconsidered(Schoderetal.2003,2005).However,thisreviewdescribesthecommonwidelyusedthermostableDNApolymerases(Table1)andtheirimportantfeaturesforPCRapplications.

A-typepolymerasesfrombacteriaThermus

ThegenusThermusbelongstothebacteriaandtotheclassofDeinococci.Thermusspeciesgrowoptimalat65–70°C,and

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ReferencesClineetal.(1996)HuangandKeohavong(1996)Takagietal.(1997)Dietrichetal.(2002)Clineetal.(1996)Kimetal.(2007)DabrowskiandKur(1998)EckertandKunkel(1990)Flamanetal.(1994)Leeetal.(2010)n.p.Parketal.(1993)Choietal.(1999)Zhengetal.(2008)Kaledinetal.(1981)Cambon-Bonavitaetal.(2000)Bonch-Osmolovskayaetal.(1996)Clineetal.(1996)Mattilaetal.(1991)DiazandSabino(1998)Flamanetal.(1994)Kimetal.(2007)Chatterjeeetal.(2002)Leeetal.(2010)Carballeiraetal.(1990)Griffithsetal.(2007)ApplMicrobiolBiotechnol(2013)97:10243–102

noitabeCunnm°iii0hhhhh%cnhmmtd0ie382355200fen12/1//9h/ir///lued-CC°CCCC14//°°C1°CCfsenlawa°°aet000°re0°°Hme5505005t75b9919119f0a199ten%dgu0Ail′tnoab7]t3:naehtttrlr%−utnnntolbnxceuuuu%lllEuv5xlno9e[BBBB59′5]]→eoosnn′//3aeood)l/′cysnsssnse3utie:eee:eoh→nvy−yyy−ynsoi/o///o//itoxoooxosl′xb5ceaNe[NNNe[NYeupton676−−=0−06n011−0.o1pit×–1]×.45na×tp−7−×(ub]06..s16001]0.cmn17.reo−2id–58i×–×−t=pt0–66sa×–ieyc510−−01ti−0−.0−0.0eracl−01116×1trnpp12ceub::raoud××−o×××−0o.×.rqrfarre672x2p8....hEre2x.fpm[1e[262e.[n1cirehettdarnn]aon858...siis714enms/–––aept..xb40p5p0er......mEk[16n0n1ylopernulaotia550007888DNAmsr7inetep)–––––cptCi20228lem°2iOxet(777776hpomD-erhBtGnssoeiuismicssseumeisosnpsycieisbrotoisunauacgiscefwutrucasssqsocoruuaosccauccmeooccccsicmacdooouccmeceorocrekooorrrrhpyheyyyhtSPTPPPTofewivervAONDe™)stn™1faoreeVesi1slemmypeDI(aleObuoqTabNopDKafwPPPTanimnnii0mm500174///CCC°°°6999AAA′′′333ooonnn///sssYeYeYe...ppp...nnn3.2–.p00...n110827––200777ssuulisnhiapmirkooodlfrbailcifsssuuummmrrreeehhhTTT)™emyzANyDr(baifTTcTnnniiihmmmhhh80003h7.5.2.1143/6//////CCCCC2/°CCC°°°°°0°°05.7550550799919919]%%0%5759t9ttt:ntnnn−ulnAuuuobu′lllxl3BBBe[B]on/oosssnsneee:e/yyy−ys///o/oooxoYeNNNe[N65−65−0−−0011011××]×4×5−9.802.03.21.3–––×–565−−9500−0−.111101:×××−.o×p365x4.....n5e[70.4–20350.....2011p.n405787–––0222577776snalsuiclaiamrmoitutiirsflaussssvuuuumacciclccrcafooaogsccncouooootommmgrmmrrrorreeegeehhhhhTTTTT)™)a™mtTnlVeU((aluoffgilmTTTTThhni55m..23hh01/142///CC°C/CC°0°°°.50055p.91999ntnulbtn..%ppAu..0′lnn93Bssseeooyyeynn/////ooossNNNYeYe660−0−0111×××..2p4p0.....2n3n200..21––.6p055.....3n21077––52508777761aAnasNtuiilliig.ohippsosplluoizseslsunuimucchrccacpecogoohtococnscotooouommmtpmmrrrerreeepehhhehhTTTTT)™lo0p5_x1fAP(NeenphitzTTTTT−4ApplMicrobiolBiotechnol(2013)97:10243–102

Table2Formulasforcalculationofthemeltingtemperature(Tm)ofoligonucleotides(summarizedinvonAhsenetal.2001).EmpiricalformulasbasedonGCcontent,lengthandtheconcentrationofmonova-lentcations[Na+].PCRistypicallyperformedin0.05Mmonovalentcationconcentration[Na+].Thenumberofnucleotidesintheoligonucle-otideisn.Mismatchesandadditiveslikedetergentsarenot

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recommended.TheempiricalresultscalculatedwiththeT7primerallowthedeductivereasoningthatoptimalannealingconditionsshouldbeoptimizedusinggradientfunctionofthermalcyclers.However,nexttothemanualwayforempiricalcalculationoftheTm,manyprogramsareinusewhichconsideradditionalsequence-specificstackingeffectsandthermodynamicdata

Tm(°C)56.053.259.051.953.151.651.351.1

Formulasforcalculatingthemeltingtemperature(Tm)ofoligonucleotidesusingtheexampleofT7primerwiththesequenceTAATACGACTCACTATAGGGand0.05Mmonovalentcationconcentration4×(G+C)+2×(A+T)69.3+0.41×(%GC)−650/n69.3+0.41×(%GC)−535/n

77.1+0.41(%GC)+11.7(log10[Na+])−528/n

77.8+0.41(%GC)+11.7(log10[Na+]×(1.0+0.7×[Na+])−1−528/n80.4+0.345(%GC)+log10[Na+]×[17.0−0.135(%GC)]−550/n81.5+0.41(%GC)+16.6(log10[Na+])−500/n

81.5+0.41(%GC)+16.6log10([Na+]×(1.0+0.7×[Na+])−1)−500/n

theysurvivetemperaturesbetween50and80°C.TheirheatstableA-typeDNApolymerasewhichbelongstothesamefamilythatincludestheprokaryoticPolIpolymerases(BraithwaiteandIto1993)hasbeenanalysedsincemanyyears(Chienetal.1976),andeversincethistimemoreandmorepolymerasesfromThermuswerecharacterized.The

Table3Thermalconductivityofdifferentmaterials.Thethermalcon-ductivitydeterminesthenecessaryenergyconsumptionthusalsothetimeuntilthereactionreachestheprogrammedtemperature.Thethermalconductivityisthecapabilitytotransportthermalenergythroughheatconduction[W=watt,m=meter,K=kelvin].Silverandaluminiumblocksareusedinthermalcyclers.PCRreactionmixturescontainnatriumorpotassiumsalts.TubesandmicrotitreplateswithdifferentthicknessareinuseMaterial

ThermalCommentsconductivity(W/mK)

ThebestmaterialbuttooexpensiveThebestmetalusedforthemoblocksUsedforheatedlids

Usedasthinoverlaytoreducetheoxidizingofsilver

ThemostcommonusedmaterialforthermoblocksandheatedlidsAcomponentofPCRbufferAcomponentofPCRbuffer

Thevolumeofasolutionhaveaninfluenceonthethermalflow,inparticularhighervolumes,e.g.100μl

Thethicknesshasadirectinfluenceofthethermalflow.Muchmorethanalowvolumesolution,e.g.10–20μl

InhibitthethermalflowandthePCR,e.g.plasticisnotcompatiblewiththeblock,orbubblesinthereactionmix

enzymeshaveamolecularmassofapproximately94–95kDa(Choietal.1999;Lawyeretal.1993;Parketal.1993),andanintrinsic5′→3′exonucleaseactivity,butlacka3′→5′proofreadingnucleaseactivity.Thefidelityofallen-zymesdependsonthepHandonconcentrationsofsubstratesMg2+anddNTPwhichformedcomplexeswithprimersandtemplates.FidelityisgoodatequimolarconcentrationsoftotaldNTPstoMg2+(Lingetal.1993).Thermuspolymerasesadda3′-adenineoverhangtoeachendofthePCRproduct.SuchPCR-amplifiedproductsareclonedintolinearizedvectorsthathavecomplementary3′-thymineoverhangs(HoltonandGraham1991;TrowerandElgar1994).ThiscloningstrategyiswellknownasTAcloning(ZhouandGomez-Sanchez2000).Commercializedkitswithpre-preparedvectorsandPCRre-agentsaregreatlysoldsincethe1990s.Overtheyears,manyDNApolymeraseswereisolatedfromdifferentThermusspe-ciesandcommercialized.

Diamond2,300Silver429Copper401Gold310Aluminium237NatriumPotassiumWater

1411350.6

Taq

Themostpopularandwell-knownthermophilicDNApoly-meraseisTaq,isolatedfromthethermophiliceubacteriumThermusaquaticus(Kaledinetal.1980).TaqwasthefirstthermostableenzymeusedforPCR(Saikietal.1988)andbecameoneofthemostimportanttoolsincommercializedmolecularbiology.Itsoptimalelongationtemperatureis75–80°C(Lawyeretal.1993),butmainlylowertemperatures(68–72°C)areusedandrecommendedbymanysuppliers,e.g.forAT-richDNA,68°Cisrecommended.ThenativeformproducedinThermusaquaticusaswellasitsrecombi-nantformproducedbyEscherichiacoliisavailable.Foroptimalelongation,divalentmagnesiumionsarenecessary(Lawyeretal.1993).MerchantsrecommendoptimizingPCRconditionsbetween1and5mMMg2+.Thehalf-lifetimeof

Plastic0.2–0.4

Airinthinlayers

0.02

10246theTaqisshortcomparedtootherpolymerasesisolatedfromArchaea:only40minat95°Cor9minat97.5°C(Lawyeretal.1993).Itisthebesttouseadenaturingtimeasshortaspossible(YuandPauls1992).And,itisrecommendedtoworkwithashortinitialstepusingarelativelylowdenaturingtemperature,e.g.94°C,ortoputtheenzymeintothereactionmixtureaftertheinitialdenaturing.Thereducedenzymeac-tivitycanbecompensatedbyextendingeachfollowingelon-gationstep.Mostthermalcyclershavethisoptionbyusingtimeincrements.Unwantedactivitywhileincreasingthetem-peratureofthefirstdenaturingstepcouldbereducedbyworkingwithhotstartTaqofferedfromdifferentmerchants.DifferentapproachesforhotstartactivationinPCRarepos-sible:blockedDNApolymeraseactivitybychemicalmodifi-cation(Birchetal.1998;Morettietal.1998),specificanti-bodies(Kellogetal.1994;Pauletal.2010;Sharkeyetal.1994),oraptamers(DangandJayasena1996)aswellasreducedactivityatlowertemperaturesbyaminoacidmutation(Kermekchievetal.2003).AnotherpossibilityforhotstartisaseparationofthemixandtheTaqbyawaxlayerwhichmeltsbetween60and80°C(Chouetal.1992).

Ingeneral,amplificationefficienciesofTaqareroundabout80%attargetsshorterthan1kbwithaCGcontentbetween45to56%(Arezietal.2003).Productyieldsgenerallydecreasewithincreasingampliconsizeabove1kb.AnovelstrategytoenhancetheprocessivityandtoimprovetheperformanceoftheTaqistofusetheTaqwithathermostableDNAbindingprotein.CovalentlinkingofthepolymerasedomainwiththeSso7dDNAbindingproteinfromSulfolobussolfataricusincreasestheprocessivity(Wangetal.2004).AsimilarproteinwhichisusedinthesamemannerisSac7dofSulfolobusacidocaldarius(Priyakumaretal.2010;Wangetal.2012).

Taqisveryoftenutilizedindiagnostics.Amplificationistotallyinhibitedinthepresenceof0.004volumepercentbloodinthePCRmixture(Al-SoudandRådström1998).Itseemsthatcontentsofhaemoglobinandlactoferrinoftheplasmainhibittheamplification.BSAwasthemostefficientamplificationfacilita-tortoincreasethistolerance(Al-SoudandRådström2001).Ca2+hasinhibitoryeffects,too.Ontheotherhand,TaqtolerateshigheramountsofcollagenthanPwo(Kimetal.2000).

AnotherdiscussedpointinhistorywasthefidelityofTaq.Ofcourse,ithasa5′→3′exonucleaseactivityandno3′→5′exonuclease(TindallandKunkel1988).Therefore,itsfidelityisnotsogoodcomparedtoPfuorVent™.However,lowfidelityconditionswereusedinthepastandpublished;theerrorratewasapproximately1–2×10−4mf×bp−1×d−1[muta-tionfrequencyperbasepairperduplication](EckertandKunkel1990;Keohavongand−5Thilly19).Highfidelityconditionsresultingin1.2×10to3.3×10−6mf×bp−1×d−1(EckertandKunkel1990;Flamanetal.1994;Lingetal.1991;Leeetal.2010)werelaterpublished.Therefore,thefidelityofTaqisbetterthanitsreputation.

ApplMicrobiolBiotechnol(2013)97:10243–102

Tfi

TfiDNApolymerasederivedfromThermusfiliformis(Choietal.1999)isalsousedinPCRapplications.Theenzymecanbeusedwithoutpayinganylicensingfee.LikeTaq,Tfihas5′→3′exonucleaseactivity.PCRperformanceiscomparabletoTaqinyield,specificity,fidelity,androbustness.Optimalextensiontemperatureis72°C,butmerchantsrecommendworkingwith68°C.TficanbesubstitutedforTaqDNApolymeraseinvirtuallyanyapplicationandisidealforgeno-mic,cDNAandplasmidtargetsupto4kb.Inaddition,TfiisfreeoflicensingrestrictionsthataccompanyTaqpolymerase.Tficanbeconsideredforuseasacomponentinadiagnosticapplicationifqualifiedbytheenduser.AmutantofTfiwhichshouldbemuchmorethermostableiscommercialized,too.Thismutantispatented(Zhengetal.2008).

TflandTth

TflandTthpolymerasesarecommercializedenzymesbutnotsowellknownasTaq.TflwasisolatedfromThermusflavus(Kaledinetal.1980;HarrellandHart1994),TthfromThermusthermophilisHB8(Carballeiraetal.1990;Rüttimannetal.1985).TflhasnearlythesamecharacteristicsasTaq(Table1).ThisappliesalsotoTth,exceptionisthehalf-lifetimewhichisshorter.TflandTthpolymerasesdisplayoptimalactivityattemperaturesbetween70and74°C.TheextensionrateofTthisroundabout1,500nucleotidesperminute,therateofTflisnotpublished.Bothenzymesaremoreresistantagainstinhibi-tionthanTaqinthepresenceofvitreouseyefluid(Wiedbrauketal.1995).Tthtolerateshigherconcentrationsofotherinhib-itorycomponentsthanTaq(Al-SoudandRådström2001).

Tbr(DyNAzyme™),TcaandHotTub™

TbrpolymerasefromThermusbrockianusismuchbetterknownunderthebrandDyNAzyme™.Tbrhasarelativelylonghalf-lifetimeof2.5hat96°CcomparedtootherpolymerasesisolatedfromThermus.Optimalamplificationconditionsareat72°Cwith1.5mMMg2+inthereaction.Marketingmaterialinformedthatamplificationupto6kbofM13DNAispossiblebutascience-basedpublicationlacks.AnotherbutnotcommercializedpolymeraseistheTcafromThermophiluscaldophilus(Parketal.1993).Itscharac-teristicsarenearlythesameasTaq(Table1)exceptitshalf-lifetimewhichis70minat95°C,muchlongerthanTaqhas.ManyDNApolymerasesfromThermusaredescribedbutitseemsthatthemarketissaturatedwithsuchenzymes.ThiscouldbeoneofthereasonswhytheHotTub™DNApolymerasefromThermusubiquitouslaunchedatthebeginningofthe1990swasdisconnected.

ApplMicrobiolBiotechnol(2013)97:10243–102A-typepolymerasesfrombacteriaThermotoga

ThepolymerasesfromThermotogawhichalsobelongstothebacteriaarenotsowellknown.Theydifferintheircharacter-isticscomparedtopolymerasesfromThermus.Onlyafewenzymesweredescribed.

Tma(UlTma™)andTne

TheTmapolymerasefromThermotogamaritimaisnotsowellknownbutwellcharacterized.ThermotogamaritimabelongstothedomainofbacteriaandtothefamilyofThermatogaceae.Itgrowsfrom50°Cupto90°Candistheonlyeubacteriumgrowingatthesehightemperatures(Huberetal.1986);onlyArchaeagrowsundercomparableconditions.TheenzymeisavailableunderthebrandUlTma™sincetheendofthe1990s.Tmapolymerasehasasizeof100kDa(ChatterjeeandPotomac1999)andaninherent3′→5′exonucleaseproofreadingactivitywithno5′→3′nucleaseactivity(DiazandSabino1998).Atruncatedformwith86kDaispatentedaswellasaformwith70kDa.Itshalf-lifetimeis50minat97.5°C(Gelfandetal.1995).ReactionconditionsandfidelityaresimilartoTaq(DiazandSabino1998)aswellastheinhibitoryeffectsofblood(Al-SoudandRådström2001).TheenzymeisalsosimilartoafewB-typepolymerasesfromArchaea.ThiswasanevidenceforalateralgenetransferbetweenArchaeaandBacteriabutageneticcheckupbetweenUlTma™andB-typeDNApolymerasescouldnotconfirmthishypothesis(HuangandIto1998).AnothersimilarpolymerasecomparedtoTmaisTne.TheenzymewasisolatedfromThermotoganeopolitanalocatedinAfricancontinentalsolfataricspring(Windbergeretal.19).TnehasthesamemolecularweightandexonucleaseactivitythanTma(Yangetal.2002).Thehalf-lifetimewastestedatverylowconditions:60minat90°C(Chatterjeeetal.2002),optimalextensiontemperatureis72–75°C.AlthoughTnewaspatented,theenzymeisnotcommerciallyavailable.

B-typeDNApolymerasesfromarchaeaThermococcalesThegeneraPyrococcusandThermococcusbelongtothedo-mainArchaea,thephylumEuryarchaeotaandtheorderThermococcales.ThehypothermophilicArchaeaandtheiren-zymesareofinterestforbiotechnologyapplication.ManyB-typeDNApolymeraseswereisolated,patentedandlaunched.Pyrococcusisathermoautotrophicarchaeon.Itsoptimalgrowthtemperatureisnearly100°Cinthedeepsea(FialaandStetter1986;Zilligetal.1987).ThemolecularmassofallisolatedB-typeDNApolymerasesfromPyrococcusisap-proximately90kDa(Takagietal.1997).Allenzymeshavea3′→5′exonucleaseactivitywhichisabletoeliminate

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misincorporatedormismatchedbasesbutnoterminaltrans-feraseactivity.Buttheydifferinhalf-lifetime,optimalreac-tionconditions,fidelitiesandelongationrates.RecombinantenzymesaremainlyproducedinE.coli,andmanyofthemarecommerciallyavailable.TheKODpolymerase,previouslyisolatedfromthestrainPyrococcussp.KOD1,belongstotheB-typeDNApolymerasesisolatedfromThermococcusbe-causephylogeneticanalysisof16SribosomalDNAhasshownitsnewphylogeneticposition(Atomietal.2004).Thermococcusisalsoahyperthermophilicmarinearchebacterium.ItbelongstotheEuryarchaeotaandgrowsoptimallyattemperaturesof80°Corhigher.Manythermo-stableB-typeDNApolymerasesarealsoisolatedandcom-mercialized.TheircharacteristicsaresimilartotheircloselyrelatedenzymesfromPyrococcus:3′→5′exonucleaseactiv-ity,no5′→3′exonucleaseactivity,molecularweightroundabout88–90kDa,lowerrorrateandlonghalf-lifetime(Table1).Inthefollowing,themajorthermophilicB-typepolymerasesaredescribed.

KOD

KODpolymerasewasisolatedfromThermococcuskodakarensisKOD1,previouslyPyrococcussp.KOD1(Atomietal.2004).ItsrecombinantenzymeproducedinE.colihasoptimalactivityatpH6.5at75°C(Takagietal.1997)butveryoften68–72°Careusedforelongation.Mg2+isoptimalbetween1.5and4.0mM.Merchantsrecommendedtovalidatein0.25mMstepsusingMgSO4.Thepolymeraseproducesblunt-endedDNAproductssuitableforcloninginbluntendligationkits.Thermostabilityis12hat95°C,resultingintheuseofhighdenaturingtempera-tures,e.g.98°C.Itselongationrateis6.0–7.8kbpermin;thisisveryfastincomparisontootherdescribedDNApolymerases(Takagietal.1997).Therefore,itcouldbeimportanttoadjusttheextensiontimeto10–25s/kbtoavoiddegradationofthePCRproduct.−V6alidationshould1bedonein5s/kbsteps.Thefidelityof2.6×10(mf×bp−×d−1)iscomparabletootherB-typepoly-merasesbutmuchbetterthanTaq.LongdistancePCRover5kbispossiblebutresultedinalossofproductyield(Nishiokaetal.2001).KODhas3′→5′exonucleaseactivity.AmixtureofKODandKOD(exo−)isdesignedforreliableamplificationoflong,complextargetsupto15kbwithrobustyieldandaccuracy.ItcanalsobeusedforincorporationofderivativedNTPs(Masudetal.2001;Nishiokaetal.2001;Sawaietal.2002).KODHotStartDNApolymerasewasoptimizedfortheamplificationofthemostdifficulttargets,e.g.90%GC-rich(Rualetal.2004);itisamixtureofKODandtwomonoclonalantibodies.TheantibodiesinhibittheexonucleaseandDNApolymeraseactivitiesatambi-enttemperatures(Mizuguchietal.1999),providinghightem-platespecificitybypreventingprimerdegradationandmisprimingeventsduringreactionset-up.KODwaslaunchedapproximately10yearslaterthanTaq.Nevertheless,itwasused

10248formanyapplications,e.g.constructionofknock-outtargetingvector(Kimetal.2005),genecloning(Herrinetal.2005;Ikeharaetal.2004),genomicDNAcloning(Nisoleetal.2004),second-strandcDNAsynthesis(Sasakietal.2004),syntheticgenesynthesis(Wuetal.2006)andmuchmore.Furthermore,poly-merasesisolatedfromThermococcuskodakarensisarealsoavail-ableunderdifferentbrands,e.g.AccuPrime™Pfx.

Tli(Vent™)

TliDNApolymerasewasisolatedfromThermococcuslitoralis(Neuneretal.1990).TheenzymehasamolecularweightofapproximatelykDaandiscommercializedandwellknownunderthebrandVent™polymerase.BothnativeanditsrecombinantformproducedinE.colihavenearlythesameactivityandfidelity(Mattilaetal.1991;Kongetal.1993).Vent™containsa3′→5′proofreadingnucleaseactiv-ityresulting1inhighfidelityof4.5×10−5–2.8×10−6−1mf×bp×d−(Carielloetal.1991;Mattilaetal.1991).Fidelityexperimentswereperformedat2mMMgSO4.Theerrorratesupto10mMMg2+areinthesamerange(Lingetal.1991);thisappliesalsoforthepHrangebetween7.0and8.5.Vent™produces95%bluntendproductsandcanbeusedforappro-priateligation.Butthe3′→5′exonucleaseactivitycouldbeproblematicfortheligationbecauseoligonucleotidesmightbedegradedattheir3′-termini.Phoshorothioatelinkagesatthe3′-endofoligonucleotidesavoidthisproblem(deNoronhaandMullins1992).Theselinkagesenhancetheprimingspec-ificity,too.TheamplificationratewithphoshorothioateprotectedprimersandusedVent™incomparisontoPfuwasdifferentiallydiscussed.Ononehand,theamplificationrateofVent™islower(Skerra1992),ontheotherhandbetterthanPfu(VigneaultandDrouin2005).Amutantwith3′→5′exonucleasenegativeactivityisalsocommercialized:Vent™(exo−).Thismutantproducesonly70%bluntendproductsand30%singlebaseoverhang(unpublisheddata).Primersarenotdegradedbutthelostnucleaseactivityresultsina40timeslowerfidelity(Mattilaetal.1991).Nevertheless,Vent™(exo−)isausefultoolforbluntendligation.Vent™hasgotcomparedtootherpolymerasesalonghalf-lifetimewhichisnearly2hat100°C.ThisismuchbetterthanTaqandalsobetterthanPfu(Kongetal.1993).Therefore,Vent™isespeciallyusefulforhightemperatureDNAsynthesisreactionconditions(Mattilaetal.1991).TheamplificationbyVent™showedahightoleranceagainstbloodcomponents(Al-SoudandRådström2001).

Pab(Isis™)

ThearchaeonPyrococcusabyssigrowsatanaerobichyperthermophilconditionsat100°Cinthedeepsea.Its

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PabDNApolymeraseishighlythermostable(Gueguenetal.2001).Pabhasa3′→5′exonucleaseactivity,whichisresponsibleforcorrectionofmismatcheddNTPs.Thehalf-lifetimeis5hat100°C,muchbetterthanPfu.ItsrecombinantformproducedinE.coliwasonthemarketunderthebrandIsis™DNApolymerase.Optimalfidelitywasmeasuredat40μMdNTPsand1.5mMMgSO4.Theerror−1rateisbetween4.7×10−6and6.6×10−7(mf×bp−1×d)dependingonMg2+anddNTPconcentration(Dietrichetal.2002).Thismeansthattheprobabilityofmisincorporatingabaseis1per1,600moleculesof1.0-kbdouble-strandedDNApercycle.ThepolymeraseisattractiveforapplicationsrequiringastrictlimitationoferrorsinPCR(Dietrichetal.2002).PabcomparedtoTaq,Pfu,VentandKODisnotsothoroughlyexplored.

Pfu

IncontrasttoPab,PfuDNApolymeraseisverywellknown.Theenzymewasisolatedfromthehyperthermo-philicmarinearchebacteriumPyrococcusfuriosus(Lundbergetal.1991).Bothitsnativeandrecombinantformsarecommerciallyavailable.Theiractivitiesarethesame(LuandErickson1997).ThemechanisticarchitectureofcrystallizedPfuwasdescribedindetail(Kennedyetal.2009).Oneofthemaincharacteristicsisthehighfidelity.Lowerrorrates,approximately1.0×10−6(mf×bp−1×d−1),wereobservedduringPCRamplificationperformedinthepresenceof2–3mMMg2+,100–300μMeachdNTP(Andréetal.1997;Clineetal.1996;Flaman1994;Lundbergetal.1991).ThepHrangebetween8.5and9.1adjustedat25°Cseemstobeoptimal.Temperaturedepen-dencyofthebufferresultedinarealvalueroundaboutpH7.1to7.7at72°C(Clineetal.1996).IncreasingMg2+concentrationsfrom2to10mMhasnoinfluenceonthefidelitybutlowerconcentrationthan2mMhaveanegativeeffect.Hotspotsofmutationsarethreetransversions:GC→TA,AT→TA,AT→CG,andonetransition:AT→GC(Andréetal.1997).Theproofreading3′→5′exonucle-aseactivityremovesmismatcheswhichisoneoftherea-sonsforitshighfidelity.Butthisadvantageisalsoadisadvantagebecauseitsnucleaseactivitydegradesfreeprimersatthe3′-end.Therefore,itisrecommendedtoworkwithnuclease-resistantphosphorothioatelinkagesatthe3′-endofoligonucleotidestoavoiddegradation(Skerra1992).Ontheotherhand,thereisaPfu(exo−)commercialized.Its3′→5′exonucleaseactivityisinactivated.Primersarenotdegradedbutthelostnucleaseactivityresultsina40timeslowerfidelity(Clineetal.1996),approximately4.7×10−5(mf×bp−1×d−1).Duringtheamplification,thePfuandPfu(exo−)donotgenerateanextranucleotideoverhang.Therefore,theproductscanbeusedforbluntligation.

ApplMicrobiolBiotechnol(2013)97:10243–102Pfu(exo−)seemstobeanattractivealternativeforahighlyefficientligation-mediatedpolymerasechainreaction(Angersetal.2001).TheamplificationwithaGCcontenthigherthan70%wassuccessfulusing20mMNaClinsteadofKCland20%dimethylsulfoxide.Moreover,extremelyGC-richDNAsequences,e.g.CGGrepeats,werealsoamplifiedbyusingPfu(Chongetal.1994).Especiallythelonghalf-lifetime(Table1)canbeusedforhightempera-turedenaturingconditions,e.g.98°C.IncontrasttotheA-typeDNApolymerasesfromThermus,theextensionrateof0.5–1.5kb×min−1islow.Elongationstepsshouldbelon-ger.Amplificationefficienciesattargetlengthsbetween2and4kbaremuchbetterthanwithTaq(Arezietal.2003).ProcessivitycouldbeenhancedbyfusingPfuwiththermo-stableSso7dDNAbindingproteinfromSulfolbussolfataricusorSac7dofS.acidocaldarius(Wangetal.2004,2012).Moreover,PfuiscommerciallyavailablemixedwithanarchaealdUTPase.IncontrasttoTaq,PfuutilizesdUTPmore.TheaccumulationofdUTPwasfoundduringPCRthroughdCTPdeamination,andthelowincor-porationofdUMPlimitedtheefficiencyofPfu(Slupphaugetal.1993).ThedUTPaseconverteddUTPtodUMPandinorganicpyrophosphate.Thecombinationofbothen-zymesresultedintheamplificationoflongertargetsandhigheryield(DabrowskiandKiaerAhring2003;Hogrefeetal.2002).Inaddition,kitswithPfuandmonoclonalantibodieswerelaunchedforhotstartresultinginanin-creaseoffidelity,specificityandthoughput.

Pst(DeepVent™)

PstDNApolymerasewasisolatedfromPyrococcusspeciesGB-Dwhichgrowsinthedeepseaat2,000m.Itisabletogrowat104°C(Jannaschetal.1992).ItsDNApolymerasewhichhasamolecularweightofkDaisonthemarketasDeepVent™.Thethermostabilityisverystrong:8hat100°Cand23hat95°C.Theextensionrateis1.4kbp×min−1.DeepVent5™isgenerallyproducedinE.coli.Thefidelityis1.2×10−–2.7×10−6(mf×bp−1×d−1)using2mMMgSO4.FidelityseemstobeinagoodrangebutitisdiscussedthatitsrelatedenzymesPfu,PaborPwoaremoreaccurate(Clineetal.1996;Flamanetal.1994).Psthasno5′→3′proofreadingexonu-cleaseactivitybut3′→5′oneresultedinaPCRproductwith95%bluntends(Table1).Amutantwithinactivated3′→5′exonucleaseactivityisonthemarket(HuangandKeohavong1996−1),whichhasalowerfidelitywith2.0×10−4(mf×bp−1×d).TheendsofPCRproductareamixofbluntandsinglebase3′overhang(Table1).MerchantsrecommendtouseDeepVent™forGC-richandloopedsequencesaswellasforprimerextension.Aspecialapplicationistheprimerextensionwithα-L-threofuranosylthymidine-3′-triphosphate1(ChaputandSzostak2003).

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Pwo

PwoDNApolymerasewasisolatedfromPyrococcuswoesei.ItsrecombinantformisproducedinE.coli(DabrowskiandKur1998;Ghasemietal.2011).Theenzymehasgotamolecularweightof90kDaandpossesses,likeallB-typepolymerases,ahigh3′→5′exonucleaseactivitywellknownasproofreadingactivity.Thereisnodetectable5′→3′exonu-cleaseactivity.Thenucleotideconcentrationshouldbeatleast200μMforeachdNTP.Lowerconcentrationsmightincreasefidelitybutmayalsoleadtodegradationofprimersandproductsbyelevated5′→3′exonucleaseactivity.Toover-comeslowdegradationofprimers,nuclease-resistantphosphorothionateprotectedprimerscouldbeused,orlongprimerswithmaximizedGCcontentmaybeadvantageous.Alternativelyhot-startshouldreducetheproblemofprimerdegradation.Thehalf-lifetimeofPwois2hat100°C.MerchantsrecommendvalidatingoptimalMg2+conditionswhichshouldbebetween1and10mM;standardis2mM.IncontrasttoTaqwhichrequiresoptimalactivityusingMgCl2,PwoshowshigheractivitywithMgSO4.PCRprod-uctsareblunt-endedandcanthereforebedirectlyusedforblunt-endligationwithoutanypretreatmentoftheends.WhenchangingfromTaqtoPwo,DNApolymeraseproblemshavebeenobserved.Merchantsrecommendreadjustingtheoptimalannealingtemperature.Amplificationwasinhibitedbybloodcomponents(Al-SoudandRådström2001)andcollagen(Kimetal.2000).

Tgo

TgoDNApolymeraseisanenzymeisolatedfromThermococcusgorganariuslivingingeothermalventsinNewZealand(Miroshnichenkoetal.1998).Theenzymehasnodetectable5′–3′exonucleaseactivity(Bonch-Osmolovskayaetal.1996)butithasgotahigh3′→5′exonucleaseactivity,alsoknownasproofreadingactivity.Theinherent3′→5′proofreadingactivityof1Tgoresultsinaverygoodfidelityupto3.5×10−6(mf×bp−1×d−).Thenucleotideconcentrationshouldbeatleast200μMforeachdNTP.Lowernucleotideconcentrationscanincreasefidel-itybutcanalsoenhancethedegradationofprimersandproducts.Furthermore,the3′→5′exonucleaseactivityofTgoalsoactsonsingle-strandedoligonucleotidesinthepresenceandabsenceofdNTPs.ThisactivitydoesusuallynotinterferewithPCRperfor-mancebutitshouldbetakenintoconsiderationforprimerdesign.Longerprimerswithphosphorothioateprotectionorwithmaxi-mizedGCcontentandfocussedcomplementarityatthe5′-endcanbeadvantageous.Fragmentsupto3.5kbcanbeamplifiedwiththestandardprotocolcontaining50mMTris/HClpH8.5,12.5mM(NH4)2SO4and35mMKCl.ItisimportanttotitratetheamountofTgo.TheoptimalconcentrationofTgovariesbetween0.4and1.25unitsperreaction,dependingonthe

10250amountoftemplatewhichcandifferfrom5ngupto200ng.Tgoexhibitsincreasedthermalstabilitywithahalf-lifeofmorethan2hat95°C(Table3).ThestandardconcentrationofMg2+is1.25mM.Variationshavealoweffectonthesensitivity(Bonch-Osmolovskayaetal.1996).Theproductsareblunt-endedandcanthereforebedirectlyusedforbluntendligationwithoutanypretreatmentoftheends.Theenzymeacceptsmodifiednucleo-tidessuchasdigoxigenin-dUTP,biotin-dUTPandfluorescein-dUTP.ForswitchingfromTaqDNApolymerasetoTgo,itseemstobebettertolowertheannealingtemperatureby2–3°Csincethe3′→5′exonucleaseactivitywillshortentheoligonucleotidesduringcycling,andtoincreasetheelongationtime.

Tfu,TNA1_pol,Tpe,TziandotherB-typepolymerasesTfuDNApolymerasewasisolatedfromThermococcusfumicolans(Cambon-Bonavitaetal.2000).Polymerizationac-tivityisoptimalat72°C,anditshalf-lifetimeat95°Cis3.3h.Measuredfidelityisquitegoodwithvaluesbetween0.9×10−55–5.3×10−(mf×bp−1×d−1).Butitselongationrateis0.32kbperminwhichisverylow.Nevertheless,productsupto10kbcouldbeamplified.However,Tfuwascommercialized.

TNA1_polpolymerasewasisolatedfromThermococcussp.NA1(Kimetal.2007).ItscharacteristicsaresimilartoVent™:90kDa,3′→5′exonucleaseactivity,optimalexten-siontemperatureat75°Candhalf-lifetimeof3.5hat100°C(Kimetal.2007).However,theextensionratewith3.6kb/minismuchbetter.Amplificationofa15-kb-longfragmentfromλDNAisdescribed,anditwasdiscussedthatthisamplificationrateisbetterthanperformedbyVent™orKODhavingalimitat8–9kb(Kimetal.2007;Mattilaetal.1991).

TpeDNApolymerasewasisolatedfromhyperthermophil-icarchaeaThermococcuspeptonophilus.OptimalPCRreac-tionconditionsarepH7at75°C,2mMMg2+,80mMKCland0.02−%6TritonX(Leeetal.2010).Thedescribedfidelityis3.37×10mf×bp−1×d−1.TpecouldamplifytargetDNAupto4kb.Ratiosof31:1ofTaqtoTpeDNApolymerasemixturesallowedPCRamplificationoftargetswithhighyieldbetween6and8kbandatamaximumupto15kb(Leeetal.2010).

TziDNApolymerasewasisolatedfromThermococcuszilligii(Griffithsetal.2007).PCRproductsupto1,600bpDNAfragmentswasamplified.TherecombinantformwaslaunchedunderthebrandPfx50™DNApolymerase.Theenzymewasfusedtoanaccessoryprotein.Thehighlyther-mostablepolymerasehasgotaproofreading3′→5′exonucle-aseactivity,whiletheaccessoryproteinstabilizesprimer–templatecomplexesinPCR.Inaddition,thefusionenzymehasanintrinsichot-startcapabilityforroomtemperaturere-actionassembly.

ApplMicrobiolBiotechnol(2013)97:10243–102

OtherpolymerasesfromThermococcusspecieswerede-scribedmoreorlesscompletely,buttheseenzymesareneitherwellknownnoravailable:Tag(Böhlkeetal.2000),Tce(Kimetal.2011),Tma(Baeetal.2009),Tpa(Ppyunetal.2012),Tthi(Marsicetal.2008)andTwa(Choetal.2012).Theseenzymesarenotdescribedinthisreview.

OtherthermostableDNApolymerases

IncontrasttothepolymerasesfromEuryarchaeota,wheresev-eralenzymesarecommercialized,afewfromCrenarchaeotaarealsocharacterized,e.g.Pyrobaculumislandicum(KählerandAntranikian2000),Pyrodictumoccultum(Uemorietal.1995),Aeropyrumpernix(Cannetal.1999),andS.solfataricus(Datukishvilietal.1996),butallofthemarewithoutanycommercialsignificance.AnextremelythermostableDNApo-lymerasefromthearchaebacteriumPyrolobusfumariusretainsfullactivityafterincubationat95°Cfor4h(CallenandMathur1999).MerchantsrecommendthisenzymeforGC-richtem-plates.AnovelfamilyarethethermostableY-DNApolymerases.TheenzymesareusedforPCRamplificationofdamagedorancientDNAs(McDonaldetal.2006).

MixturesofA-typewithB-typepolymerases

Theefficientamplificationoflongertargetsthan5kbwithonlyonepolymeraseseemstobeproblematic(Barnes1994).TheprocessivityofpolymerasescouldbeenhancedbylinkingaDNAbindingprotein,e.g.Sac7dandSso7d(Priyakumaretal.2010;Wangetal.2004,2012).AnothermethodofresolutiontoincreasetheprocessivityistheapplicationofmixtureswithA-typeandB-typepolymerases(Barnes1994).Itsuseresultedintheeffectiveamplificationoflongertargets.Asmallamountof3′→5′proofreadingenzymeincreasestheamplificationoflongertargets:Upto42kbcouldbeamplifiedwithsuchmixtures(Chengetal.1994).Activityproportionsfrom10:1to200:1ofA-typetoB-typeareusedandmustbevalidateddependingontargetandenzymes(Chengetal.1994).Manymixturesarelaunchedunderdifferentbrands.Often,itseemstobenotobvi-ouswhatisbehindthename,e.g.theHerculase™isamixtureofTaqandPfu,ortheExpand™isamixtureofTaqandPwo.

Patentsituation

Mostofthedescribedpolymerasesarepatented,afewnot.Thequestionis:WhatisthekeyofinnovationtopatentanewthermostableDNApolymerase?Istheisolationfromaneworganismenoughinnovation?Ingeneral,mostofthenewpatentedenzymesworklikethedescribedandpatentedonesbefore.Considerably,thereisnonewcreativemind,isnotit?

ApplMicrobiolBiotechnol(2013)97:10243–102Inspiteofthisblemish,moreandmoresimilarenzymeswereaccreditedaspatents.

Conclusion

TheconclusionofthisreviewisthatnothermostableDNApolymeraseisthebestone.Table1representsacompressedoverviewofthecharacteristicswell-knownthermostableDNApolymerases.Thesectionsofthespecificenzymessupplymuchmoreinformation,advantagesanddisadvan-tages.Therefore,thisminireviewshouldbeahelpfultoolforscientistsworkingwithPCR.However,theapplicationitselfreducesthechoiceofenzymes.EvaluationofoptimalconditionswillbeoftennecessaryformostPCRprocesses.

AcknowledgmentsTheauthorthanksProf.A.Steinbüchelforhissupportthisreview.

References

Al-SoudWA,RådströmP(1998)CapacityofninethermostableDNA

polymerasestomediateDNAamplificationinthepresenceofPCR-inhibitingsamples.ApplEnvironMicrobiol:3748–3753

Al-SoudWA,RådströmP(2000)Effectsofamplificationfacilitatorson

diagnosticPCRinthepresenceofblood,feces,andmeat.JClinMicrobiol38:4463–4470

Al-SoudWA,RådströmP(2001)Purificationandcharacterizationof

PCR-inhibitorycomponentsinbloodcells.JClinMicrobiol39:485–493

AndréP,KimA,KhrapkoK,ThillyWG(1997)Fidelityandmutational

spectrumofPfuDNApolymeraseonahumanmitochondrialDNAsequence.GenomeRes7:843–852

AngersM,CloutierJF,Castonguay(−)A,DrouinR(2001)Optimalcondi-tionstousePfuexoDNApolymeraseforhighlyefficientligation-mediatedpolymerasechainreactionprotocols.NucleicAcidsRes29:E83

AreziB,XingW,SorgeJA,HogrefeHH(2003)Amplificationefficiency

ofthermostableDNApolymerases.AnalBiochem321:226–235AtomiH,FukuiT,KanaiT,MorikawaM,ImanakaT(2004)Description

ofThermococcuskodacaraensissp.nov.,awellstudiedhyperther-mophilicarchaeonpreviouslyreportedasPyrococcussp.KOD1.Archaea1:263–267

BaeH,KimKP,LeeJI,SongJG,KilEJ,KimJS,KwonST(2009)

CharacterizationofDNApolymerasefromthehyperthermophilicarchaeonThermococcusmarinusanditsapplicationtoPCR.Extremophiles13:657–667

BarnesWM(1994)PCRamplificationofupto35-kbDNAwithhigh

fidelityandhighyieldfromlambdabacteriophagetemplates.ProcNatlAcadSci91:2216–2220

BirchDE,LairdWJ,ZoccoliA(1998)Nucleicacidamplificationusinga

reversiblyinactivatedthermostableenzyme.PatentapplnoUS5773258

BöhlkeK,PisaniFM,VorgiasCE,FreyB,SobekH,RossiM,

AntranikianG(2000)PCRperformanceoftheB-typeDNApoly-merasefromthethermophiliceuryarchaeonThermococcusaggregansimprovedbymutationsintheY-GG/Amotif.NucleicAcidsRes28:3910–3917

10251

Bonch-OsmolovskayaE,SvetlichnyV,AnkenbauerW,Schmitz-AgheguianG,AngererB,EbenbichlerC,LaueF(1996)Thermo-stablenucleicacidpolymerasefromThermococcusgorgonarius.PatentapplnoEP0834570A1

BraithwaiteDK,ItoJ(1993)Compilation,alignment,andphylogenetic

relationshipsofDNApolymerases.NucleicAcidsRes21:787–802CallenW,MathurEJ(1999)Isolationandidentificationofpolymerases.

PatentUS5948666A

Cambon-BonavitaMA,SchmittP,ZiegerM,FlamanJM,LesongeurF,

RaguénèsG,BindelD,FrischN,LakkisZ,DupretD,BarbierG,QuérellouJ(2000)Cloning,expression,andcharacterizationofDNApolymeraseIfromthehyperthermophilicarchaeaThermococcusfumicolans.Extremophiles4:215–225

CannIK,IshinoS,NomuraN,SakoY,IshinoY(1999)TwofamilyB

DNApolymerasesfromAeropyrumpernix,anaerobichyperther-mophiliccrenarchaeote.JBacteriol181:5984–5992

CarballeiraN,NazabalM,BritoJ,GarciaO(1990)Purificationofa

thermostableDNApolymerasefromThermusthermophilusHB8,usefulinthepolymerasechainreaction.Biotechniques9:276–281CarielloNF,SwenbergJA,SkopekTR(1991)FidelityofThermococcus

litoralisDNApolymerase(Vent)inPCRdeterminedbydenaturinggradientgelelectrophoresis.NucleicAcidsRes19:4193–4198ChangM,LeeHJ(2005)Gradientpolymerasechainreactionperfor-manceusingregularthermalcyclemachine.AnalBiochem340:174–177

ChaputJC,SzostakJW(2003)TNAsynthesisbyDNApolymerases.J

AmChemSoc125:9274–9275

ChatterjeeDK,PotomacN(1999)ClonedDNApolymerasesfrom

Thermotogamaritimaandmutantsthereof.PatentapplnoUS5948614

ChatterjeeDK,PotomacN,HughesJ(2002)ClonedDNApolymerases

fromThermotoganeopolitana.PatentapplnoUS44424B1ChengS,FocklerC,BarnesWM,HiguchiR(1994)Effectiveamplifica-tionoflongtargetsfromclonedinsertsandhumangenomicDNA.ProcNatlAcadSci91:5695–5699

ChesterN,MarshakDR(1993)Dimethylsulfoxide-mediated

primerTmreduction:amethodforanalyzingtheroleofrenaturationtemperatureinthepolymerasechainreaction.AnalBiochem209:284–290

ChienA,EdgarDB,TrelaJM(1976)Deoxyribonucleicacidpolymerase

fromtheextremethermophileThermusaquaticus.JBact127:1550–1557

ChoSS,KimKP,LeeKK,YounMH,KwonST(2012)Characterization

andPCRapplicationofanewhigh-fidelityDNApolymerasefromThermococcuswaiotapuensis.EnzymeMicrobTechnol51:334–341

ChoiJJ,JungSE,KimHK,KwonST(1999)Purificationandproperties

ofThermusfiliformisDNApolymeraseexpressedinEscherichiacoli.BiotechnolApplBiochem30:19–25

ChongSS,EichlerEE,NelsonDL,HughesMR(1994)Robustamplifi-cationandethidium-visibledetectionofthefragileXsyndromeCGGrepeatusingPfupolymerase.AmJMedGenet51:522–526ChouQ,RussellM,BirchDE,RaymondJ,BlochW(1992)

Preventionofpre-PCRmis-primingandprimerdimerizationimproveslow-copy-numberamplifications.NucleicAcidsRes20:1717–1723

ClineJ,BramanJC,HogrefeHH(1996)PCRfidelityofpfuDNA

polymeraseandotherthermostableDNApolymerases.NucleicAcidsRes24:36–3551

DabrowskiS,KiaerAhringB(2003)Cloning,expression,andpurifica-tionoftheHis6-taggedhyper-thermostabledUTPasefromPyrococcuswoeseiinEscherichiacoli:applicationinPCR.ProteinExprPurif31:72–78

DabrowskiS,KurJ(1998)CloningandexpressioninEscherichiacoliof

therecombinanthis-taggedDNApolymerasesfromPyrococcusfuriosusandPyrococcuswoesei.ProteinExprPurif14:131–138

10252

DangC,JayasenaSD(1996)OligonucleotideinhibitorsofTaqDNA

polymerasefacilitatedetectionoflowcopynumbertargetsbyPCR.JMolBiol2:268–278

DatukishviliN,PokholokD,LottspeichF,PrangishviliD,

RechinskyV(1996)TheDNApolymerase-encodinggenefromathermoacidophilicarchaeonSulfolobusacidocaldarius.Gene177:271–273

deNoronhaCM,MullinsJI(1992)Amplimerswith3′-terminal

phosphorothioatelinkagesresistdegradationbyventpolymeraseandreduceTaqpolymerasemispriming.PCRMethodsAppl2:131–136

DiazRS,SabinoEC(1998)Accuracyofreplicationinthepolymerase

chainreaction.ComparisonbetweenThermotogamaritimaDNApolymeraseandThermusaquaticusDNApolymerase.BrazJMedBiolRes31:1239–1242

DietrichJ,SchmittP,ZiegerM,PreveB,RollandJL,ChaabihiH,

GueguenY(2002)PCRperformanceofthehighlythermostableproof-readingB-typeDNApolymerasefromPyrococcusabyssi.FEMSMicrobiolLett217:–94

EckertKA,KunkelTA(1990)HighfidelityDNAsynthesisbytheThermus

aquaticusDNApolymerase.NucleicAcidsRes18:3739–3744

FialaG,StetterKO(1986)Pyrococcusfuriosussp.nov.representsa

novelgenusofmarineheterotrophicarchaebacteriagrowingopti-mallyat100°C.ArchMicrobiol145:56–61

FlamanJM,FrebourgT,MoreauV,CharbonnierF,MartinC,IshiokaC,

FriendSH,IggoR(1994)ArapidPCRfidelityassay.NucleicAcidsRes22:3259–3260

GelfandGH,LawyerFC,StoffelS(1995)Mutatedthermostablenucleic

acidpolymeraseenzymefromThermotogamaritima.PatentapplnoUS20029

GhasemiA,SalmanianAH,SadeghifardN,SalarianAA,GholiMK

(2011)Cloning,expressionandpurificationofPwopolymerasefromPyrococcuswoesei.IranJMicrobiol3:118–122

GriffithsK,NayakS,ParkK,MandelmanD,ModrellB,LeeJ,NgB,

GibbsMD,BergquistPL(2007)NewhighfidelitypolymerasesfromThermococcusspecies.ProteinExprPurif52:19–30

GueguenY,RollandJL,LecompteO,AzamP,LeRomancerG,Flament

D,RaffinJP,DietrichJ(2001)CharacterizationoftwoDNApoly-merasesfromthehyperthermophiliceuryarchaeonPyrococcusabyssi.EurJBiochem268:5961–5969

HarrellRA2nd,HartRP(1994)RapidpreparationofThermusflavus

DNApolymerase.PCRMethodsAppl3:372–375

HenkeW,HerdelK,JungK,SchnorrD,LoeningSA(1997)Betaine

improvesthePCRamplificationofGC-richDNAsequences.NucleicAcidsRes25:3957–3958

HerrinBR,GroegerAL,JustementLB(2005)TheadaptorproteinHSH2

attenuatesapoptosisinresponsetoligationoftheBcellantigenreceptorcomplexontheBlymphomacellline,WEHI-231.JBiolChemm280:3507–3515

HogrefeHH,HansenCJ,ScottBR,NielsonKB(2002)Archaeal

dUTPaseenhancesPCRamplificationswitharchaealDNApoly-merasesbypreventingdUTPincorporation.ProcNatlAcadSci99:596–601

HoltonTA,GrahamMW(1991)Asimpleandefficientmethodfordirect

cloningofPCRproductsusingddT-tailedvectors.NucleicAcidsRes19:1156

HuangYP,ItoJ(1998)ThehyperthermophilicbacteriumThermotoga

maritimahastwodifferentclassesoffamilyCDNApolymerases:evolutionaryimplications.NucleicAcidsRes26:5300–5309

HuangH,KeohavongP(1996)Fidelityandpredominantmutationsproduced

bydeepventwild-typeandexonuclease-deficientDNApolymerasesduringinvitroDNAamplification.DNACellBiol15:5–594

HuberR,LangworthyTA,KönigH,ThommM,WoeseCR,SleytrUB,

StetterKO(1986)Thermotogamaritimasp.nov.representsanewgenusofuniqueextremelythermophiliceubacteriagrowingupto90°C.ArchMicrobiol144:324–333

ApplMicrobiolBiotechnol(2013)97:10243–102

IkeharaY,IkeharaSK,PaulsonJC(2004)NegativeregulationofTcell

receptorsignalingbySiglec-7(p70/AIRM)andSiglec-9.JBiolChem279:43117–43125

JannaschHW,WirsenCO,MolyneauxSJ,LangworthyTA(1992)Com-parativephysiologicalstudiesonhyperthermophilicarchaeaisolatedfromdeep-seahotventswithemphasisonPyrococcusstrainGB-D.ApplEnvironMicrobiol58:3472–3481

KählerM,AntranikianG(2000)Cloningandcharacterizationofafamily

BDNApolymerasefromthehyperthermophiliccrenarchaeonPyrobaculumislandicum.JBacteriol182:655–663

KaledinAS,SliusarenkoAG,GorodetskiĭSI(1980)Isolationandprop-ertiesofDNApolymerasefromextremethermophylicbacteriaThermusaquaticusYT-1.Biokhimiia45:4–651,Russian

KellogDE,RybalkinI,ChenS,MukhamedovaN,VlasikT,SiebertPD,

ChenchikA(1994)TaqStartAntibody:“hotstart”PCRfacilitatedbyaneutralizingmonoclonalantibodydirectedagainstTaqDNApolymerase.Biotechniques16:1134–1137

KennedyEM,HergottC,DewhurstS,KimB(2009)Themechanistic

architectureofthermostablePyrococcusfuriosusfamilyBDNApolymerasemotifAanditsinteractionwiththedNTPsubstrate.Biochemistry48:11161–11168

KeohavongP,ThillyWG(19)FidelityofDNApolymerasesinDNA

amplification.ProcNatlAcadSci86:9253–9257

KermekchievMB,TzekovA,BarnesWM(2003)Cold-sensitivemutants

ofTaqDNApolymeraseprovideahotstartforPCR.NuclAcidsRes31:6139–6147

KimS,LabbeRG,RyuS(2000)Inhibitoryeffectsofcollagenonthe

PCRfordetectionofClostridiumperfringens.ApplEnvironMicrobiol66:1213–1215

KimTS,MaedaA,MaedaT,HeinleinC,KedishviliN,PalczewskiK,

NelsonPS(2005)Delayeddarkadaptationin11-cis-retinoldehydrogenase-deficientmice:aroleofRDH11invisualprocessesinvivo.JBiolChem280:8694–8704

KimYJ,LeeHS,BaeSS,JeonJH,LimJK,ChoY,NamKH,KangSG,Kim

SJ,KwonST,LeeJH(2007)Cloning,purification,andcharacterizationofanewDNApolymerasefromahyperthermophilicarchaeon,Thermococcussp.NA1.JMicrobiolBiotechnol17:1090–1097

KimKP,BaeH,KimIH,KwonST(2011)Cloning,expression,andPCR

applicationofDNApolymerasefromthehyperthermophilicarchaeon,Thermococcusceler.BiotechnolLett33:339–346

KongH,KuceraRB,JackWE(1993)CharacterizationofaDNA

polymerasefromthehyperthermophilearchaeaThermococcuslitoralis.VentDNApolymerase,steadystatekinetics,thermalsta-bility,processivity,stranddisplacement,andexonucleaseactivities.JBiolChem268:1965–1975

LawyerFC,StoffelS,SaikiRK,ChangSY,LandrePA,AbramsonRD,

GelfandDH(1993)High-levelexpression,purification,andenzy-maticcharacterizationoffull-lengthThermusaquaticusDNApo-lymeraseandatruncatedformdeficientin5′to3′exonucleaseactivity.PCRMethodsAppl2:275–287

LeeJI,KimYJ,BaeH,ChoSS,LeeJ,KwonS(2010)Biochemical

propertiesandPCRperformanceofafamilyBDNApolymerasefromhyperthermophilicEuryarchaeonThermococcuspeptonophilus.ApplBiochemBiotech160:1585–1599

LingLL,KeohavongP,DiasC,ThillyWG(1991)Optimizationofthe

polymerasechainreactionwithregardtofidelity:modifiedT7,Taq,andVentDNApolymerases.PCRMethodsAppl1:63–69

LuC,EricksonHP(1997)ExpressioninEscherichiacoliofthethermo-stableDNApolymerasefromPyrococcusfuriosus.ProteinExprPurif11:179–184

LundbergKS,ShoemakerDD,AdamsMW,ShortJM,SorgeJA,Mathur

EJ(1991)High-fidelityamplificationusingathermostableDNApolymeraseisolatedfromPyrococcusfuriosus.Gene108:1–6

MarsicD,FlamanJM,NgJD(2008)NewDNApolymerasefromthe

hyperthermophilicmarinearchaeonThermococcusthioreducens.Extremophiles12:775–788

ApplMicrobiolBiotechnol(2013)97:10243–102

MasudMM,Ozaki-NakamuraA,SatouF,OhbayashiT,OzakiH,Sawai

H(2001)EnzymaticsynthesisofmodifiedDNAbyPCR.NucleicAcidsResSuppl1:21–22

MattilaP,KorpelaJ,TenkanenT,PitkänenK(1991)FidelityofDNA

synthesisbytheThermococcuslitoralisDNApolymerase—anextremelyheatstableenzymewithproofreadingactivity.NucleicAcidsRes19:4967–4973

McDonaldJP,HallA,GasparuttoD,CadetJ,BallantyneJ,WoodgateR

(2006)NovelthermostableY-familypolymerases:applicationsforthePCRamplificationofdamagedorancientDNAs.NucleicAcidsRes34:1102–1111

MiroshnichenkoML,GongadzeGM,RaineyFA,KostyukovaAS,Lysenko

AM,ChernyhNA,Bonch-OsmolovskayaEA(1998)Thermococcusgorgonariussp.nov.andThermococcuspacificussp.nov.:heterotro-phicextremelythermophilicarchaeafromNewZealandsubmarinehotvents.IntJSystBacteriol48:23–29

MizuguchiH,NakatsujiM,FujiwaraS,TakagiM,ImanakaT(1999)

CharacterizationandapplicationtohotstartPCRofneutralizingmono-clonalantibodiesagainstKODDNApolymerase.Biochem126:762–768

MorettiT,KoonsB,BudowleB(1998)EnhancementofPCRamplifica-tionyieldandspecificityusingAmpliTaqGoldDNApolymerase.Biotechniques25:716–722

NeunerA,JannaschHW,BelkinS,StetterKO(1990)Thermococcus

litoralissp.nov.:anewspeciesofextremelythermophilicmarinearchaebacteria.ArchMicrobiol153:205–207

NishiokaM,MizuguchiH,FujiwaraS,KomatsubaraS,KitabayashiM,

UemuraH,TakagiM,ImanakaT(2001)LongandaccuratePCRwithamixtureofKODDNApolymeraseanditsexonucleasedeficientmutantenzyme.JBiotechnol88:141–149

NisoleS,LynchC,StoyeJP,YapMW(2004)ATrim5-cyclophilinA

fusionproteinfoundinowlmonkeykidneycellscanrestrictHIV-1.ProcNatlAcadSci101:13324–13328

OwczarzyR,MoreiraBG,YouY,BehlkeMA,WalderJA(2008)

PredictingstabilityofDNAduplexesinsolutionscontainingmag-nesiumandmonovalentcations.Biochemistry47:5336–5353

ParkJH,KimJS,KwonST,LeeDS(1993)Purificationandcharacteri-zationofThermuscaldophilusGK24DNApolymerase.EurJBiochem214:135–140

PaulN,ShumJ,LeT(2010)HotstartPCR.MethodsMolBiol630:301–318PpyunH,KimI,ChoSS,SeoKJ,YoonK,KwonST(2012)ImprovedPCR

performanceusingmutantTpa-SDNApolymerasesfromthehyperther-mophilicarchaeonThermococcuspacificus.JBiotechnol1:363–370PriyakumarUD,RamakrishnaS,NagarjunaKR,ReddySK(2010)

Structuralandenergeticdeterminantsofthermalstabilityandhier-archicalunfoldingpathwaysofhyperthermophilicproteins,Sac7dandSso7d.JPhysChemB114:1707–1718

ReesWA,YagerTD,KorteJ,vonHippelPH(1993)Betainecan

eliminatethebasepaircompositiondependenceofDNAmelting.Biochemistry32:137–144

RualJF,Hirozane-KishikawaT,HaoT,BertinN,LiS,DricotA,LiN,

RosenbergJ,LameschP,VidalainPO,ClingingsmithTR,HartleyJL,EspositoD,CheoD,MooreT,SimmonsB,SequerraR,BosakS,Doucette-StammL,LePeuchC,VandenhauteJ,CusickME,AlbalaJS,HillDE,VidalM(2004)HumanORFeomeversion1.1:aplatformforreverseproteomics.GenomeRes14:2128–2135

RüttimannC,CotorásM,ZaldívarJ,VicuñaR(1985)DNApolymerases

fromtheextremelythermophilicbacteriumThermusthermophilusHB-8.EurJBiochem149:41–46

SaikiRK,GelfandDH,StoffelS,ScharfSJ,HiguchiR,HornGT,Mullis

KB,ErlichHA(1988)Primer-directedenzymaticamplificationofDNAwithathermostableDNApolymerase.Science239:487–491SarkarG,KapelnerS,SommerSS(1990)Formamidecandramatically

improvethespecificityofPCR.NucleicAcidsRes18:7465

SasakiY,CasolaS,KutokJL,RajewskyK,Schmidt-SupprianM(2004)

TNFfamilymemberBcell-activatingfactor(BAFF)receptor-

10253

dependentand-independentrolesforBAFFinBcellphysiology.JImmunol173:2245–2252

SawaiH,Ozaki-NakamuraA,MineM,OzakiH(2002)Synthesisofnew

modifiedDNAsbyhyperthermophilicDNApolymerase:substrateandtemplatespecificityoffunctionalizedthymidineanaloguesbear-ingansp3-hybridizedcarbonattheC5alpha-positionforseveralDNApolymerases.BioconjugChem13:309–316

SchoderD,SchmalwieserA,SchaubergerG,KuhnM,HoorfarJ,Wagner

M(2003)Physicalcharacteristicsofsixnewthermocyclers.ClinChem49:960–963

SchoderD,SchmalwieserA,SchaubergerG,HoorfarJ,KuhnM,Wagner

M(2005)NovelapproachforassessingperformanceofPCRcyclersusedfordiagnostictesting.JClinMicrobiol43:2724–2728

SharkeyDJ,ScaliceER,ChristyKGJr,AtwoodSM,DaissJL(1994)

Antibodiesasthermolabileswitches:hightemperaturetriggeringforthepolymerasechainreaction.Biotechnology12:506–509

SkerraA(1992)Phosphorothioateprimersimprovetheamplificationof

DNAsequencesbyDNApolymeraseswithproofreadingactivity.NucleicAcidsRes20:3551–35

SlupphaugG,AlsethI,EftedalI,VoldenG,KrokanHE(1993)Low

incorporationofdUMPbysomethermostableDNApolymerasesmaylimittheiruseinPCRamplifications.AnalBiochem211:1–169TakagiM,NishiokaM,KakiharaH,KitabayashiM,InoueH,Kawakami

B,OkaM,ImanakaT(1997)CharacterizationofDNApolymerasefromPyrococcussp.StrainKOD1anditsapplicationtoPCR.ApplEnvironMicrobiol63:4504–4510

TindallKR,KunkelTA(1988)FidelityofDNAsynthesisbytheThermus

aquaticusDNApolymerase.Biochemistry27:6008–6013

TrowerMK,ElgarGS(1994)PCRcloningusingT-vectors.Methods

MolBiol31:19–33

UemoriT,IshinoY,DoiH,KatoI(1995)Thehyperthermophilic

archaeonPyrodictiumoccultumhastwoalpha-likeDNApolymer-ases.JBacteriol177:21–2177

VaradarajK,SkinnerDM(1994)Denaturantsorcosolventsimprovethe

specificityofPCRamplificationofaG+C-richDNAusinggenet-icallyengineeredDNApolymerases.Gene140:1–5

VigneaultF,DrouinR(2005)Optimalconditionsandspecific

characteristicsofVentexo−DNApolymeraseinligation-mediatedpolymerasechainreactionprotocols.BiochemCellBiol83:147–165

vonAhsenN,WittwerCT,SchützE(2001)Oligonucleotidemelting

temperaturesunderPCRconditions:nearest-neighborcorrectionsforMg(2+),deoxynucleotidetriphosphate,anddimethylsulfoxideconcentrationswithcomparisontoalternativeempiricalformulas.ClinChem47:1956–1961

WangY,ProsenDE,MeiL,SullivanJC,FinneyM,VanderHornPB

(2004)AnovelstrategytoengineerDNApolymerasesforenhancedprocessivityandimprovedperformanceinvitro.NucleicAcidsRes32:1197–1207

WangY,HornPV,XiL(2012)Methodsofusingimprovedpolymerases.

USpatent20120295270

WiedbraukDL,WernerJC,DrevonAM(1995)InhibitionofPCRby

aqueousandvitreousfluids.JClinMicrobiol33:23–26

WindbergerE,HuberR,TrinconeA,FrickeR,StetterK(19)

Thermotogathermarumsp.nov.andThermotoganeapolitanaoc-curringinAfricancontinentalsolfataricsprings.ArchMicrobiol151:506–512

WuDY,UgozzoliL,PalBK,QianJ,WallaceRB(1991)Theeffectof

temperatureandoligonucleotideprimerlengthonthespecificityandefficiencyofamplificationbythepolymerasechainreaction.DNACellBiol10:233–238

WuG,WolfJB,IbrahimAF,VadaszS,GunasingheM,FreelandSJ

(2006)Simplifiedgenesynthesis:aone-stepapproachtoPCR-basedgeneconstruction.JBiotechnol124:496–503

YangSW,AstatkeM,PotterJ,ChatterjeeDK(2002)Mutant

ThermotoganeapolitanaDNApolymeraseI:alteredcatalytic

102

propertiesfornon-templatednucleotideadditionandincorpo-rationofcorrectnucleotides.NucleicAcidsRes30:4314–4320

YuK,PaulsKP(1992)OptimizationofthePCRprogramforRAPD

analysis.NucleicAcidsRes20:2606

ZhangZ,YangX,MengL,LiuF,ShenC,YangW(2009)Enhanced

amplificationofGC-richDNAwithtwoorganicreagents.Biotechniques47:775–779

ApplMicrobiolBiotechnol(2013)97:10243–102

ZhengW,LeeJE,PotterRJ,MandelmanD(2008)DNApolymerase

blendsandmutantDNApolymerases.Patentapplno200802525ZhouMY,Gomez-SanchezCE(2000)UniversalTAcloning.CurrIssues

MolBiol2:1–7

ZilligW,HolzI,KlenkHP,TrentJ,WunderlS,JanekovicD,ImselE,HaasB

(1987)Pyrococcuswoesei,sp.nov.,anultra-thermophilicmarinear-chaebacterium,representinganovelorder,Thermococcales.SystemAppMicrobiol9:62–70