酶切位点保护碱基.doc

《酶切位点保护碱基.doc》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。

1、CleavageClosetotheEndofDNAFragments (oligonucleotides)Totestthevaryingrequirementsrestrictionendonucleaseshaveforthenumberofbasesflankingtheirrecognitionsequences,aseriesofshort,double-strandedoligonucleotidesthatcontaintherestrictionendonucleaserecog

2、nitionsites(showninred)weredigested.Thisinformationmaybehelpfulwhenchoosingtheorderofadditionoftworestrictionendonucleasesforadoubledigest(aparticularconcernwhencleavingsitesclosetogetherinapolylinker),orwhenselectingenzymesmostlikelytocleaveattheendo

3、faDNAfragment.Theexperimentwasperformedasfollows:0.1A260unitofoligonucleotidewasphosphorylatedusingT4polynucleotidekinaseandg-[32P]ATP.1µgof5´[32P]-labeledoligonucleotidewasincubatedat20°Cwith20unitsofrestrictionendonucleaseinabuffercontaining70mMTris

4、-HCl(pH7.6),10mMMgCl2,5mMDTTandNaClorKCldependingonthesaltrequirementofeachparticularrestrictionendonuclease.Aliquotsweretakenat2hoursand20hoursandanalyzedby20%PAGE(7Murea).Percentcleavagewasdeterminedbyvisualestimateofautoradiographs.Asacontrol,self-

5、ligatedoligonucleotideswerecleavedefficiently.Decreasedcleavageefficiencyforsomeofthelongerpalindromicoligonucleotidesmaybecausedbytheformationofhairpinloops.

6、A

7、B

8、C

9、E

10、H

11、K

12、M

13、N

14、P

15、S

16、X

17、 EnzymeOligoSequenceChainLength%Cleavage2hr20hrAccIGGTCGACCCGGTCGACCGC

18、CGGTCGACCGG81012000000AflIIICACATGTGCCACATGTGGCCCACATGTGGG810120>90>900>90>90AscIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA81012>90>90>90>90>90>90AvaICCCCGGGGCCCCCGGGGGTCCCCCGGGGGA8101250>90>90>90>90>90BamHICGGATCCGCGGGATCCCGCGCGGATCCGCG8101210>90>9025>90>90BglII

19、CAGATCTGGAAGATCTTCGGAAGATCTTCC81012075250>90>90BssHIIGGCGCGCCAGGCGCGCCTTTGGCGCGCCAA81012005000>90BstEIIGGGT(A/T)ACCC9010BstXIAACTGCAGAACCAATGCATTGGAAAACTGCAGCCAATGCATTGGAACTGCAGAACCAATGCATTGGATGCAT22242702525050>90ClaICATCGATGGATCGATCCCATCGATGGCCCATCG

20、ATGGG88101200>905000>9050EcoRIGGAATTCCCGGAATTCCGCCGGAATTCCGG81012>90>90>90>90>90>90HaeIIIGGGGCCCCAGCGGCCGCTTTGCGGCCGCAA81012>90>90>90>90>90>90HindIIICAAGCTTGCCAAGCTTGGCCCAAGCTTGGG8101200100075KpnIGGGTACCCGGGGTACCCCCGGGGTACCCCG810120>90>900>90>