酶切位点保护碱基

CleavageClosetotheEndofDNAFragments(oligonucleotides)Totestthevaryingrequirementsrestrictionendonucleaseshaveforthenumberofbasesflankingtheirrecognitionsequences,aseriesofshort,double-strandedoligonucleotidesthatcontaintherestrictionendonucleaserecognitionsites(showninred)weredigested.Thisinformationmaybehelpfulwhenchoosingtheorderofadditionoftworestrictionendonucleasesforadoubledigest(aparticularconcernwhencleavingsitesclosetogetherinapolylinker),orwhenselectingenzymesmostlikelytocleaveattheendofaDNAfragment.Theexperimentwasperformedasfollows:0.1A260unitofoligonucleotidewasphosphorylatedusingT4polynucleotidekinaseand-[32P]ATP.1µgof5´[32P]-labeledoligonucleotidewasincubatedat20°Cwith20unitsofrestrictionendonucleaseinabuffercontaining70mMTris-HCl(pH7.6),10mMMgCl2,5mMDTTandNaClorKCldependingonthesaltrequirementofeachparticularrestrictionendonuclease.Aliquotsweretakenat2hoursand20hoursandanalyzedby20%PAGE(7Murea).Percentcleavagewasdeterminedbyvisualestimateofautoradiographs.Asacontrol,self-ligatedoligonucleotideswerecleavedefficiently.Decreasedcleavageefficiencyforsomeofthelongerpalindromicoligonucleotidesmaybecausedbytheformationofhairpinloops.|A|B|C|E|H|K|M|N|P|S|X|EnzymeOligoSequenceChainLength%Cleavage2hr20hrAccIGGTCGACCCGGTCGACCGCCGGTCGACCGG81012000000AflIIICACATGTGCCACATGTGGCCCACATGTGGG810120909009090AscIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA81012909090909090AvaICCCCGGGGCCCCCGGGGGTCCCCCGGGGGA81012509090909090BamHICGGATCCGCGGGATCCCGCGCGGATCCGCG81012109090259090BglIICAGATCTGGAAGATCTTCGGAAGATCTTCC810120752509090BssHIIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA8101200500090BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT2224270252505090ClaICATCGATGGATCGATCCCATCGATGGCCCATCGATGGG881012009050009050EcoRIGGAATTCCCGGAATTCCGCCGGAATTCCGG81012909090909090HaeIIIGGGGCCCCAGCGGCCGCTTTGCGGCCGCAA81012909090909090HindIIICAAGCTTGCCAAGCTTGGCCCAAGCTTGGG8101200100075KpnIGGGTACCCGGGGTACCCCCGGGGTACCCCG810120909009090MluIGACGCGTCCGACGCGTCG810025050NcoICCCATGGGCATGCCATGGCATG814050075NdeICCATATGGCCCATATGGGCGCCATATGGCGGGGTTTCATATGAAACCCGGAATTCCATATGGAATTCCGGGAATTCCATATGGAATTCCC810121820220000757500009090NheIGGCTAGCCCGGCTAGCCGCTAGCTAGCTAG810120101002550NotITTGCGGCCGCAAATTTGCGGCCGCTTTAAAATATGCGGCCGCTATAAAATAAGAATGCGGCCGCTAAACTATAAGGAAAAAAGCGGCCGCAAAAGGAAAA1216202428010102525010109090NsiITGCATGCATGCACCAATGCATTGGTTCTGCAGTT122210909090PacITTAATTAAGTTAATTAACCCTTAATTAAGG8101200002590PmeIGTTTAAACGGTTTAAACCGGGTTTAAACCCAGCTTTGTTTAAACGGCGCGCCGG8101224000750255090PstIGCTGCAGCTGCACTGCAGTGCAAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT8142224260109090001090900PvuICCGATCGGATCGATCGATTCGCGATCGCGA81012010002510SacICGAGCTCG81010SacIIGCCGCGGCTCCCCGCGGGGA812050090SalIGTCGACGTCAAAAGGCCATAGCGGCCGCGCGTCGACGTCTTGGCCATAGCGGCCGCGGACGCGTCGACGTCGGCCATAGCGGCCGCGGAA2830320101005075ScaIGAGTACTCAAAAGTACTTTT81210752575SmaICCCGGGCCCCGGGGCCCCCGGGGGTCCCCCGGGGGA68101200109010105090SpeIGACTAGTCGGACTAGTCCCGGACTAGTCCGCTAGACTAGTCTAG810121410100090905050SphIGGCATGCCCATGCATGCATGACATGCATGCATGT81214001002550StuIAAGGCCTTGAAGGCCTTCAAAAGGCCTTTT81012909090909090XbaICTCTAGAGGCTCTAGAGCTGCTCTAGAGCACTAGTCTAGACTAG810121409075750909090XhoICCTCGAGGCCCTCGAGGGCCGCTCGAGCGG810120101002575XmaICCCCGGGGCCCCCGGGGGCCCCCCGGGGGGTCCCCCCGGGGGGA810121402550900759090CleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/µg)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransformantsfromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%.BasePairsfromEndreferstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedoverhangfromtheinitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesrefertothenumberofindependenttrialsforeachenzymetested(fromMoreira,R.andNoren,C.(1995),Biotechniques,19,56-59).Note:Asageneralrule,enzymesnotlistedbelowrequire6basespairsoneithersideoftheirrecognitionsitetocleaveefficiently.|A|B|E|H|K|M|N|P|S|X|EnzymeBasepairsfromEnd%CleavageEfficiencyVectorInitialCutAatII32188(2)100(2)95(2)LITMUS29LITMUS28LITMUS29NcoINcoIPinAIAcc65I2199(2)75(3)LITMUS29pNEB193SpeISacIAflII113(2)LITMUS29StuIAgeI11100(1)100(2)LITMUS29LITMUS29XbaIAatIIApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI197(2)LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI21100(1)8(2)LITMUS39LITMUS38BsrGIBsrGIBsrGI2199(2)88(2)LITMUS39LITMUS38SphIBspEIBssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI111100(