酶切位点保护碱基

CleavageClosetotheEndofDNAFragments(oligonucleotides)Totestthevaryingrequirementsrestrictionendonucleaseshaveforthenumberofbasesflankingtheirrecognitionsequences,aseriesofshort,double-strandedoligonucleotidesthatcontaintherestrictionendonucleaserecognitionsites(showninred)weredigested.Thisinformationmaybehelpfulwhenchoosingtheorderofadditionoftworestrictionendonucleasesforadoubledigest(aparticularconcernwhencleavingsitesclosetogetherinapolylinker),orwhenselectingenzymesmostlikelytocleaveattheendofaDNAfragment.Theexperimentwasperformedasfollows:0.1Aunitofoligonucleotidewas260phosphorylatedusingT4polynucleotidekinasea-n[d32P]ATP.1gof5[32P]-labeledoligonucleotidewasincubatedaCtw2i0t°h20unitsofrestrictionendonucleaseinabuffercontaining70mMTris-HCl(pH7.6),10mM,Mg5ClmMDTTandNaClorKCldependingonthesaltrequirementofeachparticularrestrictionendonuclease.Aliquotsweretakenat2hoursand20hoursandanalyzedby20%PAGE(7Murea).Percentcleavagewasdeterminedbyvisualestimateofautoradiographs.Asacontrol,self-ligatedoligonucleotideswerecleavedefficiently.Decreasedcleavageefficiencyforsomeofthelongerpalindromicoligonucleotidesmaybecausedbytheformationofhairpinl.oops|abCEHKILINPSX|EnzymeOligoSequenceChainLength%Cleavage2hr20hrAccIGGTCGACC800CGGTCGACCG1000CCGGTCGACCGG1200AflIIICACATGTG800CCACATGTGG10>90>90CCCACATGTGGG12>90>90AscIGGCGCGCC8>90>90AGGCGCGCCT10>90>90TTGGCGCGCCAA12>90>90AvaICCCCGGGG850>90CCCCCGGGGG10>90>90TCCCCCGGGGGA12>90>90BamHICGGATCCG81025CGGGATCCCG10>90>90CGCGGATCCGCG12>90>90BglIICAGATCTG800GAAGATCTTC1075>90GGAAGATCTTCC1225>90BssHIIGGCGCGCC800AGGCGCGCCT1000TTGGCGCGCCAA1250>90BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGG2200AAAACTGCAGCCAATGCATTGGAA242550CTGCAGAACCAATGCATTGGATGCAT2725>90ClaICATCGATG800GATCGATC800CCATCGATGG10>90>90CCCATCGATGGG125050EcoRIGGAATTCC8>90>90CGGAATTCCG10>90>90CCGGAATTCCGG12>90>90HaeIIIGGGGCCCC8>90>90AGCGGCCGCT10>90>90TTGCGGCCGCAA12>90>90HindIIICAAGCTTG800CCAAGCTTGG1000CCCAAGCTTGGG121075KpnIGGGTACCC800GGGGTACCCC10>90>90CGGGGTACCCCG12>90>90MluIGACGCGTC800CGACGCGTCG102550NcoICCCATGGG800CATGCCATGGCATG145075NdeICCATATGG800CCCATATGGG1000CGCCATATGGCG1200GGGTTTCATATGAAACCC1800GGAATTCCATATGGAATTCC2075>90GGGAATTCCATATGGAATTCCC2275>90NheIGGCTAGCC800CGGCTAGCCG101025CTAGCTAGCTAG121050NotITTGCGGCCGCAA1200ATTTGCGGCCGCTTTA161010AAATATGCGGCCGCTATAAA201010ATAAGAATGCGGCCGCTAAACTAT242590AAGGAAAAAAGCGGCCGCAAAAGGAAAA2825>90NsiITGCATGCATGCA1210>90CCAATGCATTGGTTCTGCAGTT22>90>90PacITTAATTAA800GTTAATTAAC10025CCTTAATTAAGG120>90PmeIGTTTAAAC800GGTTTAAACC10025GGGTTTAAACCC12050AGCTTTGTTTAAACGGCGCGCCGG2475>90PstIGCTGCAGC800TGCACTGCAGTGCA141010AACTGCAGAACCAATGCATTGG22>90>90AAAACTGCAGCCAATGCATTGGAA24>90>90CTGCAGAACCAATGCATTGGATGCAT2600PvuICCGATCGG800ATCGATCGAT101025TCGCGATCGCGA12010SacICGAGCTCG81010SacIIGCCGCGGC800TCCCCGCGGGGA1250>90SalIGTCGACGTCAAAAGGCCATAGCGGCCGC2800GCGTCGACGTCTTGGCCATAGCGGCCGCGG301050ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA321075ScaIGAGTACTC81025AAAAGTACTTTT127575SmaICCCGGG6010CCCCGGGG8010CCCCCGGGGG101050TCCCCCGGGGGA12>90>90SpeIGACTAGTC810>90GGACTAGTCC1010>90CGGACTAGTCCG12050CTAGACTAGTCTAG14050SphIGGCATGCC800CATGCATGCATG12025ACATGCATGCATGT141050StuIAAGGCCTT8>90>90GAAGGCCTTC10>90>90AAAAGGCCTTTT12>90>90XbaICTCTAGAG800GCTCTAGAGC10>90>90TGCTCTAGAGCA1275>90CTAGTCTAGACTAG1475>90XhoICCTCGAGG800CCCTCGAGGG101025CCGCTCGAGCGG121075XmaICCCCGGGG800CCCCCGGGGG102575CCCCCCGGGGGG1250>90TCCCCCCGGGGGGA14>90>90CleavageClosetotheEndofDNAFragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/匹)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransformantsfromthedigestionreactionbythenumberobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%."BasePairsfromEnd"referstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedoverhangfromtheinitialcut.Sinceithasnotbeendemonstratedwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesrefertothenumberofindependenttrialsforeachenzymetested(fromMoreira,R.andNoren,C.(1995),Biotechniques,19,56-59).Note:Asageneralrule,enzymesnotlistedbelowrequire6basespairsoneithersideoftheirrecognitionsitetocleaveefficiently.|A|B|EIHIK|MIN|P|SIX|EnzymeBasepairsfromEnd%CleavageEfficiencyVectorInitialCutAatII388(2)LITMUS29NcoI2100(2)LITMUS28NcoI195(2)LITMUS29PinAIAcc65I299(2)LITMUS29SpeI175⑶pNEB193SacIAflII113(2)LITMUS29StuIAgeI1100(1)LITMUS29XbaI1100(2)LITMUS29AatIIApaI2100(1)LITMUS38SpeIAscI197(2)pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI197(2)LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI2100(1)LITMUS39BsrGI18(2)LITMUS38BsrGIBsrGI299(2)LITMUS39SphI188(2)LITMUS38BspEIBssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI1100(1)LITMUS29XhoI188(1)LITMUS29PstI1100(1)LITMUS39NheIEcoRV1100(2)LITMUS29PstIHindIII390(2)LITMUS29NcoI291⑵LITMUS28NcoI10(2)LITMUS29BamHIKasI297(1)LITMUS38NgoMIV193(1)LITMUS38HindIIIKpnI2100(2)LITMUS29SpeI2100(2)LITMUS29SacI199(2)pNEB193SacIMluI299(2)LITMUS39EagIMunI2100(1)LITMUS39NgoMIVNeoI2100(1)LITMUS28HindIIINgoMIV2100(1)LITMUS39MunINheI1100(1)LITMUS39EcoRI282(1)LITMUS39EagINotI7100(2)BluescriptSK-SpeI4100(1)BluescriptSK-KspI198(2)BluescriptSK-XbaINsiI3100(2)LITMUS29BssHII377⑷LITMUS29BglII295(2)LITMUS28BssHIIPaeI176⑶pNEB193BamHIPmeI194(2)pNEB193PstIPstI398(1)LITMUS29EcoRV250(5)LITMUS39HindIII137⑶LITMUS29EcoRISaeI199(2)LITMUS29AvrIISalI389(2)LITMUS39SpeI223(2)LITMUS39SphI161(3)LITMUS38SphISpeI2100(2)LITMUS29Acc65I2100(2)LITMUS29KpnISphI299(1)LITMUS39SalI297(1)LITMUS39BsrGI192(2)LITMUS38SalIXbaI199(2)LITMUS29AgeI194(1)LITMUS29PinAIXhoI197(2)LITMUS29EcoRIXmaI298(1)pNEB193AscI292(1)pNEB193BssHII