Description: PhageRNApolymerasesarewidelyusedfortheinvitrosynthesisofRNAtranscriptsfromDNAtemplateswhichhaveadouble-strandedpromoter(atleast19bases)upstreamofthesequencetobetranscribed.T7phageRNApolymerasehasahighspecificityforitsrespectivepromoter.OnceT7RNApolymerasebindstoitsdouble-strandedDNApromoter,itseparatesthetwoDNAstrands,andusesthe3'to5'strandasatemplatetosynthesizeacomplementarystrandattheendoftheDNAtemplate(run-offtranscription)(Figure1).Theinitiationoftranscriptionisarate-limitingstepin in vitrotranscriptreactions.Elongationofthetranscriptisveryfastandefficient. Cloningvectorsusuallycontaintwoormoreseparatephagepromotersflankingamultiplecloningsite.Soeitherstrandofthetemplatecanbetranscribedfromthepromoterontheoppositestrand.TheT7HighYield in vitroTranscriptionKitcanalsobeusedtotranscribefromDNAtemplatesproducedviaPCRorsyntheticDNAoligonucleotides. BecauseRNA-RNAandRNA-DNAhybridsaremorestablethanDNA-DNAduplexesinsolidbasehybridizations,andsingle-strandedRNAprobesarenotdepletedbyrehybridizationtoacomplementaryprobestrand,theT7HighYield in vitroTranscriptionKitcanbeusedtoincorporateisotopicallylabeledaswellasnonisotopicallymodifiednucleotidesintoRNAtranscriptsasprobesforhybridizationreactions,suchasNorthernandSouthernblotting,slotordotblotting,insituhybridizations,andnucleaseprotectionassays.IftheRNAproducedwillbeusedasaprobetodetectmRNA,itisimportantthatmRNA-complementary(antisense)transcriptsaresynthesized.Withcustomsuppliedcapanalog,fullyfunctionalcappedmRNAscanbemadethroughT7HighYield in vitroTranscriptionreactions.
Application: -Invitrotranscription -Invitrotranslation -Hybridizationprobesgeneration -RNaseprotectionassays -RNAbindingproteinassays -RNAistudy
Reference: SchendornETandMierindorfRC(1985).NucleicAcidsRes,13,6223-6236 |