Applications

• Ligationamplification(LigaseChainReaction,LCR)^1-7
• RepeatExpansionDetection(RED)⁷
• Simultaneousmutagenesisofmultiplesites^8,9
• Otherligation-baseddetectionmethods

Ampligase®ThermostableDNALigasecatalyzesNAD-dependentligationofadjacent3´-hydroxylatedand5´-phosphorylatedterminiinduplexDNAstructuresthatarestableathightemperatures.Derivedfromathermophilicbacterium,theenzymeisstableandactiveatmuchhighertemperaturesthanconventionalDNAligases.Itshalf-lifeis48hoursat65°Candgreaterthan1 hourat95°C.AmpligaseDNALigasehasbeenshowntobeactiveforatleast500thermalcycles(94°C/80°C)or16hoursofcycling.¹⁰Thisexceptionalthermostabilitypermitsextremelyhighhybridizationstringencyandligationspecificity.AmpligaseDNALigasehasnodetectableactivityinligatingblunt-endedDNAandhasnoactivityonRNAorRNA:DNAhybrids.

UnitDefinition:OneunitofAmpligaseDNALigasecatalyzestheligationof50%ofthecossitesin1µgoflamBDaDNAin1minuteat45°Cin1XAmpligaseReactionBuffer.

Note:OneunitofAmpligaseDNALigaseisequivalenttoatleast15ofthe"cohesiveendunits"or"nickligationunits"definedelsewhere.⁴

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

Ampligase10XReactionBuffer:200mMTris-HCl(pH8.3),250mMKCl,100mMMgCl₂,5mMNAD,and0.1%Triton®X-100.

QualityControl:AmpligaseThermostableDNALigaseisassayedfortheabsenceofblunt-endligationactivitybyligatingSmaI-digestedbacteriophagelambdaDNAat45°C;ligationoccursonlyatcossitesasdeterminedbygelelectrophoresis.AmpligaseDNALigaseisfreeofdetectableexo-andendonucleaseandRNaseactivities.

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