酶切位點(diǎn)保護(hù)堿基

1、CleavageClosetotheEndofDNAFragments(oligonucleotides)Totestthevaryingrequirementsrestrictionendonucleaseshaveforthenumberofbasesflankingtheirrecognitionsequences,aseriesofshort,double-strandedoligonucleotidesthatcontaintherestrictionendonucleaserecognitionsites(showninred)weredigested.Thisinformatio

2、nmaybehelpfulwhenchoosingtheorderofadditionoftworestrictionendonucleasesforadoubledigest(aparticularconcernwhencleavingsitesclosetogetherinapolylinker),orwhenselectingenzymesmostlikelytocleaveattheendofaDNAfragment.Theexperimentwasperformedasfollows:0.1A260unitofoligonucleotidewasphosphorylatedusing

3、T4polynucleotidekinaseand-32PATP.1pgof532P-labeledoligonucleotidewasincubatedat20Cwith20unitsofrestrictionendonucleaseinabuffercontaining70mMTris-HCl(pH7.6),10mMMgCl2,5mMDTTandNaClorKCldependingonthesaltrequirementofeachparticularrestrictionendonuclease.Aliquotsweretakenat2hoursand20hoursandanalyzed

4、by20%PAGE(7Murea).Percentcleavagewasdeterminedbyvisualestimateofautoradiographs.Asacontrol,self-ligatedoligonucleotideswerecleavedefficiently.Decreasedcleavageefficiencyforsomeofthelongerpalindromicoligonucleotidesmaybecausedbytheformationofhairpinloops.Al旦ce凹KIMN1PIS1X1EnzymeOligoSequenceChainLengt

5、h%Cleavage2hr20hrAccIGGTCGACC800CGGTCGACCG1000CCGGTCGACCGG1200AflIIICACATGTG800CCACATGTGG109090CCCACATGTGGG129090AscIGGCGCGCC89090AGGCGCGCCT109090TTGGCGCGCCAA129090AvaICCCCGGGG85090CCCCCGGGGG109090TCCCCCGGGGGA129090BamHICGGATCCG81025CGGGATCCCG109090CGCGGATCCGCG129090BglIICAGATCTG800GAAGATCTTC107590G

6、GAAGATCTTCC122590BssHIIGGCGCGCC800AGGCGCGCCT1000TTGGCGCGCCAA125090BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGG2200AAAACTGCAGCCAATGCATTGGAA242550CTGCAGAACCAATGCATTGGATGCAT272590ClaICATCGATG800GATCGATC800CCATCGATGG109090CCCATCGATGGG125050EcoRIGGAATTCC89090CGGAATTCCG109090CCGGAATTCCGG129090HaeIII

7、GGGGCCCC89090AGCGGCCGCT109090TTGCGGCCGCAA129090HindIIICAAGCTTG800CCAAGCTTGG1000CCCAAGCTTGGG121075KpnIGGGTACCC800GGGGTACCCC109090CGGGGTACCCCG129090MluIGACGCGTC800CGACGCGTCG102550NcoICCCATGGG800CATGCCATGGCATG145075NdeICCATATGG800CCCATATGGG1000CGCCATATGGCG1200GGGTTTCATATGAAACCC1800GGAATTCCATATGGAATTCC2

8、07590GGGAATTCCATATGGAATTCCC227590NheIGGCTAGCC800CGGCTAGCCG101025CTAGCTAGCTAG121050NotITTGCGGCCGCAA1200ATTTGCGGCCGCTTTA161010AAATATGCGGCCGCTATAAA201010ATAAGAATGCGGCCGCTAAACTAT242590AAGGAAAAAAGCGGCCGCAAAAGGAAAA282590NsiITGCATGCATGCA121090CCAATGCATTGGTTCTGCAGTT229090PacITTAATTAA800GTTAATTAAC10025CCTTAA

9、TTAAGG12090PmeIGTTTAAAC800GGTTTAAACC10025GGGTTTAAACCC12050AGCTTTGTTTAAACGGCGCGCCGG247590PstIGCTGCAGC800TGCACTGCAGTGCA141010AACTGCAGAACCAATGCATTGG229090AAAACTGCAGCCAATGCATTGGAA249090CTGCAGAACCAATGCATTGGATGCAT2600PvuICCGATCGG800ATCGATCGAT101025TCGCGATCGCGA12010SacICGAGCTCG81010SacIIGCCGCGGC800TCCCCGCG

10、GGGA125090SalIGTCGACGTCAAAAGGCCATAGCGGCCGC2800GCGTCGACGTCTTGGCCATAGCGGCCGCGG301050ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA321075ScaIGAGTACTC81025AAAAGTACTTTT127575SmaICCCGGG6010CCCCGGGG8010CCCCCGGGGG101050TCCCCCGGGGGA129090SpeIGACTAGTC81090GGACTAGTCC101090CGGACTAGTCCG12050CTAGACTAGTCTAG14050SphIGGCATGCC800C

11、ATGCATGCATG12025ACATGCATGCATGT141050StuIAAGGCCTT89090GAAGGCCTTC109090AAAAGGCCTTTT129090XbaICTCTAGAG800GCTCTAGAGC109090TGCTCTAGAGCA127590CTAGTCTAGACTAG147590XhoICCTCGAGG800CCCTCGAGGG101025CCGCTCGAGCGG121075XmaICCCCGGGG800CCCCCGGGGG102575CCCCCCGGGGGG125090TCCCCCCGGGGGGA149090CleavageClosetotheEndofDNA

12、Fragments(linearizedvector)Linearizedvectorswereincubatedwiththeindicatedenzymes(10units/匹)for60minutesattherecommendedincubationtemperatureandNEBufferforeachenzyme.Followingligationandtransformation,cleavageefficienciesweredeterminedbydividingthenumberoftransformantsfromthedigestionreactionbythenum

13、berobtainedfromreligationofthelinearizedDNA(typically100-500colonies)andsubtractingfrom100%.BasePairsfromEndreferstothenumberofdouble-strandedbasepairsbetweentherecognitionsiteandtheterminusofthefragment;thisnumberdoesnotincludethesingle-strandedoverhangfromtheinitialcut.Sinceithasnotbeendemonstrate

14、dwhetherthesesingle-strandednucleotidescontributetocleavageefficiency,4basesshouldbeaddedtotheindicatednumberswhendesigningPCRprimers.Averageefficiencieswereroundedtothenearestwholenumber;experimentalvariationwastypicallywithin10%.Thenumbersinparenthesesrefertothenumberofindependenttrialsforeachenzy

15、metested(fromMoreira,R.andNoren,C.(1995),Biotechniques,19,56-59).Note:Asageneralrule,enzymesnotlistedbelowrequire6basespairsoneithersideoftheirrecognitionsitetocleaveefficiently.|A|B|E|H|K|M|N|P|S|X|EnzymeBasepairsfromEnd%CleavageEfficiencyVectorInitialCutAatII388(2)LITMUS29NcoI2100(2)LITMUS28NcoI19

16、5LITMUS29PinAIAcc65I299(2)LITMUS29SpeI175pNEB193SacIAflII113LITMUS29StuIAgeI1100(1)LITMUS29XbaI1100(2)LITMUS29AatIIApaI2100(1)LITMUS38SpeIAscI197pNEB193BamHIAvrII1100(2)LITMUS29SacIBamHI197LITMUS29HindIIIBglII3100(2)LITMUS29NsiIBsiWI2100(2)LITMUS29BssHIIBspEI2100(1)LITMUS39BsrGI18(2)LITMUS38BsrGIBsr

17、GI299(2)LITMUS39SphI188(2)LITMUS38BspEIBssHII2100(2)LITMUS29BsiWIEagI2100(2)LITMUS39NheIEcoRI1100(1)LITMUS29XhoI188(1)LITMUS29PstI1100(1)LITMUS39NheIEcoRV1100(2)LITMUS29PstIHindIII390(2)LITMUS29NcoI291LITMUS28NcoI10(2)LITMUS29BamHIKasI297(1)LITMUS38NgoMIV193(1)LITMUS38HindIIIKpnI2100(2)LITMUS29SpeI2

18、100(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)LITMUS29BssHII377LITMUS29BglII295LITMUS28BssHIIPaeI176pNEB193BamHIPmeI194(2)pNEB193PstIPstI398(1)LITMUS29EcoRV250(5)LITMUS39HindIII137LITMUS29E