Description: Duetoitsthermostablenature, Thermotoganeapolitana DNApolymerase(Tne)wasidentified(1)asanenzymeabletowithstandtheprotein-denaturingconditions(hightemperature)requiredduringPCR(2).SimilartoE.coliDNApolymeraseI,butunlikeTaqDNApolymerase,TneDNApolymerasecontainsboth3'->5'and5'->3'-exonuleaseactivities.Therefore, itreplacedtheDNApolymerasefromE.colioriginallyusedinPCR(3).Taq'soptimumtemperatureforactivityis75-80°C,withahalf-lifeof9minutesat97.5°C,andcanreplicatea1000bpstrandofDNAinlessthan10secondsat72°C(4). TheDNAproductshaveanA(adenine)overhangsattheir3'ends.ThismaybeusefulinTAcloning,wherebyacloningvector(suchasaplasmid)thathasaT(thymine)3'-overhangisused,whichcomplementswiththeAoverhangofthePCRproduct,enablingligationofthePCRproductintotheplasmidvector.
Application: -PCR(ordinaryandhigh-throughput) -PrimerExtension -MicroarrayAnalysis -Denaturinghighperformanceliquidchromatography(DHPLC)
Source: Thermotoganeapolitana(Tne)DNApolymerasebelongstotheDNApolymeraseI(PolI)family.
RecommendedReactionConditions: 94°C,1minute.->(94°C,10seconds.->55°C,30seconds.->72°C,30seconds.)for25cycles.
RecommendedStorageCondition:-20ºC
References: 1.ChienA,EdgarDB,TrelaJM(1976)."DeoxyribonucleicacidpolymerasefromtheextremethermophileThermusaquaticus".J.Bact.127(3):1550–7.PMC232952.PMID8432. 2.Saiki,RK;etal.(1988)."Primer-directedenzymaticamplificationofDNAwithathermostableDNApolymerase.".Science239(4839):487–91.doi:10.1126/science.2448875.PMID2448875. 3.Saiki,RK;etal.(1985)."Enzymaticamplificationofbeta-globingenomicsequencesandrestrictionsiteanalysisfordiagnosisofsicklecellanemia".Science230(4732):1350–doi:10.1126/science.2999980.PMID2999980. 4.LawyerFC,etal.(1993)."High-levelexpression,purification,andenzymaticcharacterizationoffull-lengthThermusaquaticusDNApolymerase...".PCRMethodsAppl.2(4):275–87.PMID8324500. |