HighlythermostableDNAligaseforapplicationswhereligationathightemperatureisbeneficial

  • Epicentre ProductHighthermostabilityallowsligationusinghigh-stringencyhybridizationconditions
  • HighspecificityandstringencypermitssensitivedetectionofSNPs11

Applications

  • Ligationamplification(LigaseChainReaction,LCR)1-7
  • RepeatExpansionDetection(RED)7
  • Simultaneousmutagenesisofmultiplesites8,9
  • 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.

References

  1. Landegren,U.etal.(1988)Science242,229.
  2. Wu,D.Y.andWallace,R.B.(1989)Genomics4,560.
  3. Barany,F.(1991)Proc.Natl.Acad.Sci.USA88,189.
  4. Birkenmeyer,L.andArmstrong,A.S.(1992)J.Clin.Micro.30,3089.
  5. Dille,B.J.etal.(1993)J.Clin.Micro.31,729
  6. Nakazawa,H.etal.(1994)Proc.Natl.Acad.Sci.USA91,360.
  7. Sirugo,G.andKidd,K.(1995)EpicentreForum2(3),1.
  8. Moore,D.andMichael,S.(1995)EpicentreForum2(4),4.
  9. Rouwendal,G.J.etal.(1993)BioTechniques15,68.
  10. Schalling,M.etal.(1993)NatureGenetics4,135.
  11. Hardenbol,P. etal. (2003) NatureBiotechnology 21,673.

ORDERINFORMATION

Ampligase™isprovidedatastandardconcentrationof5U/μl,withtheexceptionofcatalognumbersA0102KandA110K(100U/μl).

AmpligaseDNALigaseKitcontains:AmpligaseDNALigase,10XReactionBuffer,andControlDNA.AmpligaseEnzyme&Buffercontain:AmpligaseDNALigaseand10XReactionBuffer(25μlBufferforeach50Uenzyme).

OneunitofAmpligase™isequaltoasmanyas15unitsofotherthermostableDNAligases.Pleasecomparecompetitiveunitdefinitions.