酶切位点所加保护碱基

寡核苷酸近末端位点的酶切(CleavageClosetotheEndofDNAFragments(oligonucleotides))为了解不同内切酶对识别位点以外最少保护碱基数目的要求，NEB采用了一系列含识别序列的短双链寡核苷酸作为酶切底物进行实验。实验结果对于确定双酶切顺序将会有帮助（比如在多接头上切割位点很接近时），或者当切割位点靠近DNA末端时也很有用。在本表中没有列出的酶，则通常需在识别位点两端至少加上6个保护碱基，以确保酶切反应的进行。实验方法：用γ-[32P]ATP在T4多聚核苷酸激酶的作用下标记0.1A260单位的寡核苷酸。取1µg已标记了的寡核苷酸与20单位的内切酶，在20°C条件下分别反应2小时和20小时。反应缓冲液含70mMTris-HCl(pH7.6),10mMMgCl2,5mMDTT及适量的NaCl或KCl（视酶的具体要求而定）。20%的PAGE（7M尿素）凝胶电泳分析，经放射自显影确定酶切百分率。本实验采用自连接的寡核苷酸作为对照。若底物有较长的回文结构，切割效率则可能因为出现发夹结构而降低。酶寡核苷酸序列链长切割率%2hr20hrAccIGGTCGACCCGGTCGACCGCCGGTCGACCGG81012000000AflIIICACATGTGCCACATGTGGCCCACATGTGGG810120909009090AscIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA81012909090909090AvaICCCCGGGGCCCCCGGGGGTCCCCCGGGGGA81012509090909090BamHICGGATCCGCGGGATCCCGCGCGGATCCGCG81012109090259090BglIICAGATCTGGAAGATCTTCGGAAGATCTTCC810120752509090BssHIIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA8101200500090BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT2224270252505090ClaICATCGATGGATCGATCCCATCGATGGCCCATCGATGGG881012009050009050EcoRIGGAATTCCCGGAATTCCGCCGGAATTCCGG81012909090909090HaeIIIGGGGCCCCAGCGGCCGCTTTGCGGCCGCAA81012909090909090HindIIICAAGCTTGCCAAGCTTGGCCCAAGCTTGGG8101200100075KpnIGGGTACCCGGGGTACCCCCGGGGTACCCCG810120909009090MluIGACGCGTCCGACGCGTCG810025050NcoICCCATGGGCATGCCATGGCATG814050075NdeICCATATGGCCCATATGGGCGCCATATGGCGGGGTTTCATATGAAACCCGGAATTCCATATGGAATTCCGGGAATTCCATATGGAATTCCC810121820220000757500009090NheIGGCTAGCCCGGCTAGCCGCTAGCTAGCTAG810120101002550NotITTGCGGCCGCAAATTTGCGGCCGCTTTAAAATATGCGGCCGCTATAAAATAAGAATGCGGCCGCTAAACTATAAGGAAAAAAGCGGCCGCAAAAGGAAAA1216202428010102525010109090NsiITGCATGCATGCACCAATGCATTGGTTCTGCAGTT122210909090PacITTAATTAAGTTAATTAACCCTTAATTAAGG8101200002590PmeIGTTTAAACGGTTTAAACCGGGTTTAAACCCAGCTTTGTTTAAACGGCGCGCCGG8101224000750255090PstIGCTGCAGCTGCACTGCAGTGCAAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT8142224260109090001090900PvuICCGATCGGATCGATCGATTCGCGATCGCGA81012010002510SacICGAGCTCG81010SacIIGCCGCGGCTCCCCGCGGGGA812050090SalIGTCGACGTCAAAAGGCCATAGCGGCCGCGCGTCGACGTCTTGGCCATAGCGGCCGCGGACGCGTCGACGTCGGCCATAGCGGCCGCGGAA2830320101005075ScaIGAGTACTCAAAAGTACTTTT81210752575SmaICCCGGGCCCCGGGGCCCCCGGGGGTCCCCCGGGGGA68101200109010105090SpeIGACTAGTCGGACTAGTCCCGGACTAGTCCG8101210100909050CTAGACTAGTCTAG14050SphIGGCATGCCCATGCATGCATGACATGCATGCATGT81214001002550StuIAAGGCCTTGAAGGCCTTCAAAAGGCCTTTT81012909090909090XbaICTCTAGAGGCTCTAGAGCTGCTCTAGAGCACTAGTCTAGACTAG810121409075750909090XhoICCTCGAGGCCCTCGAGGGCCGCTCGAGCGG810120101002575XmaICCCCGGGGCCCCCGGGGGCCCCCCGGGGGGTCCCCCCGGGGGGA810121402550900759090克隆PCR产物的方法之一，是在PCR产物两端设计一定的限制酶切位点，经酶切后克隆至用相同酶切的载体中。但实验证明，大多数限制酶对裸露的酶切位点不能切断。必须在酶切位点旁边加上一个至几个保护碱基，才能使所定的限制酶对其识别位点进行有效切断。下表列举了15种限制酶，分别比较了各种限制酶在其酶切位点旁边分别加0、1、2、3个保护碱基后的切断情况。表中的：(-)为不能切断；(±)为不能完全切断；(+)为能完全切断。结果显示，基本上所有限制酶，在其酶切位点旁边加上3个以上的保护碱基后，可以对其酶切位点进行有效切断。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(1)88(1)100(1)LITMUS29LITMUS29LITMUS39XhoIPstINheIEcoRV1100(2)LITMUS29PstIHindIII32190(2)91(2)0(2)LITMUS29LITMUS28LITMUS29NcoINcoIBamHIKasI2197(1)93(1)LITMUS38LITMUS38NgoMIVHindIIIKpnI221100(2)100(2)99(2)LITMUS29LITMUS29pNEB193SpeISacISacIMluI299(2)LITMUS39EagIMunI2100(1)LITMUS39NgoMIVNcoI2100(1)LITMUS28HindIIINgoMIV2100(1)LITMUS39MunINheI12100(1)82(1)LITMUS39LITMUS39EcoRIEagINotI741100(2)100(1)98(2)BluescriptSK-BluescriptSK-BluescriptSK-SpeIKspIXbaINsiI33100(2)77(4)LITMUS29LITMUS29BssHIIBglII295(2)LITMUS28BssHIIPacI176(3)pNEB193BamHIP