Lucigen/T7 R&DNA™ Polymerase/D7P9205K/5,000 Units

T7R&DNA™Polymerase*isarecombinantmutantformofT7RNApolymerase(Y639Fmutant),inwhichTyr-639hasbeenreplacedbyPhe.^1,2ThismutationenablesT7R&DNAPolymerasetoincorporate2´-deoxyribonucleosidetriphosphates(dNTPs)intofull-lengthtranscriptsmuchmoreefficientlythanthecorrespondingwild-typeenzyme,¹whileretainingthesamecatalyticactivityforincorporationofcanonicalNTPs,andthesamehighpromoterspecificityaswild-typeT7RNAPolymerase.Theabilityofthismutantenzymetoincorporatecertain2´-modified-dNTPs(includingbutnotlimitedtodNTPshavinga2´-fluorineor2´-aminogroup)enablesefficientinvitrosynthesisoftranscriptscomposedofmixedrNMP/2´-dNMPorrNMP/2´-modified-dNMPfromanyDNAtemplatethatisdownstreamoftheT7RNApolymerasepromoter.Suchmodifiedtranscriptsareusefulformanyapplications.

Note:Completesubstitutionofone2´-dNTP(orone2´-modifieddNTP)foracanonicalrNTPinaT7R&DNAPolymerasereactionresultsinaslightdecreaseinyield.Additionalsubstitutionsof2´-dNTPs(or2´-modified-dNTPs)forcanonicalrNTPswillfurtherreduceyieldofthetranscriptproduced.Substitutionwithallfour2´-dNTPs(or2´-modifieddNTPs)willresultinextremelylowyieldsoftranscriptandisNOTRECOMMENDED.

UnitDefinition:OneunitofT7R&DNAPolymerasecatalyzestheincorporationof1nmolofribonucleosidetriphosphateintoRNAin1hourat37°CunderstandardassayconditionsusingaDNAtemplatewiththeT7promoter.

StorageBuffer:50%glycerolcontaining50mMTris-HCl(pH7.5),0.1MNaCl,1.0mMDTT,0.1mMEDTA,and0.1%Triton®X-100.

5XTranscriptionBuffer:0.2MTris-HCl(pH7.5),50mMNaCl,30mMMgCl₂,and10mMspermidine.

References

1. Sousa,R.andPadilla,R.(1995)EMBOJ.14,4609.
2. U.S.PatentNos.5,849,546and6,107,037.

*Coveredbyissuedand/orpendingpatents.