实验十一DNA酶切.ppt

实验十一实验十一的酶切的酶切RestrictionEndonucleases:AnOverviewwRestrictionenzymeswerediscoveredabout30yearsagoduringinvestigationsintothephenomenonofhost-specificrestrictionandmodificationofbacterialviruses.wBacteriainitiallyresistinfectionsbynewviruses,andthisrestrictionofviralgrowthstemmedfromendonucleaseswithinthecellsthatdestroyforeignDNAmolecules.AmongthefirstoftheserestrictionenzymestobepurifiedwereEcoRIandEcoRIIfromEscherichia coli,andHindIIandHindIIIfromHaemophilus influenzae.TheseenzymeswerefoundtocleaveDNAatspecificsites,generatingdiscrete,gene-sizefragmentsthatcouldbere-joinedinthelaboratory.wResearcherswerequicktorecognizethatrestrictionenzymesprovidedthemwitharemarkablenewtoolforinvestigatinggeneorganization,functionandexpression.Astheuseofrestrictionenzymesspreadamongmolecularbiologistsinthelate1970s,companiessuchasNewEnglandBiolabsbegantosearchformore.Exceptforcertainviruses,restrictionenzymeswerefoundonlywithinprokaryotes.wManythousandsofbacteriaandarchaehavenowbeenscreenedfortheirpresence.Analysisofsequencedprokaryoticgenomesindicatesthattheyarecommon-allfree-livingbacteriaandarchaeaappeartocodeforthem.Restrictionenzymesareexceedinglyvaried;theyrangeinsizefromthediminutivePvuII(157aminoacids)tothegiantCjeI(1250aminoacids)andbeyond.Amongover3,000activitiesthathavebeenpurifiedandcharacterized,morethan250differentsequence-specificitieshavebeendiscovered.Ofthese,over30%werediscoveredandcharacterizedatNewEnglandBiolabs.wThesearchfornewspecificitiescontinues,bothbiochemically,bytheanalysisofcell-extracts,andcomputationally,bytheanalysisofsequencedgenomes.Althoughmostactivitiesencounteredtodayturnouttobeduplicates-isoschizomers-ofexistingspecificities,restrictionenzymeswithnewspecificitiesarefoundwithregularity.Beginningintheearly1980s,NewEnglandBiolabsembarkedonaprogramtocloneandoverexpressthegenesforrestrictionenzymes.Cloningimprovesenzymepuritybyseparatingenzymesfromcontaminatingactivitiespresentinthesamecells.Italsoimprovesenzymeyieldsandgreatlysimplifiespurification,anditprovidesthegenesforsequencingandanalysis,andtheproteinsforx-raycrystallography.wRestrictionenzymesprotectbacteriafrominfectionsbyviruses,anditisgenerallyacceptedthatthisistheirroleinnature.Theyfunctionasmicrobialimmunesystems.WhenastrainofE.colilackingarestrictionenzymeisinfectedwithavirus,mostvirusparticlescaninitiateasuccessfulinfection.Whenthesamestraincontainsarestrictionenzyme,however,theprobabilityofsuccessfulinfectionplummets.Thepresenceofadditionalenzymeshasamultiplicativeeffect;acellwithfourorfiveindependentrestrictionenzymescouldbevirtuallyimpregnable.wRestrictionenzymesusuallyoccurincombinationwithoneortwomodificationenzymes(DNA-methyltransferases)thatprotectthecellsownDNAfromcleavagebytherestrictionenzyme.wModificationenzymesrecognizethesameDNAsequenceastherestrictionenzymethattheyaccompany,butinsteadofcleavingthesequence,theymethylateoneofthebasesineachoftheDNAstrands.ThemethylgroupsprotrudeintothemajorgrooveofDNAatthebindingsiteandpreventtherestrictionenzymefromactinguponit.wTogether,arestrictionenzymeanditscognatemodificationenzyme(s)formarestriction-modification(R-M)system.wInsomeR-Msystemstherestrictionenzymeandthemodificationenzyme(s)areseparateproteinsthatactindependentlyofeachother.wInothersystems,thetwoactivitiesoccurasseparatesubunits,orasseparatedomains,ofalarger,combined,restriction-and-modificationenzyme.wRestrictionenzymesaretraditionallyclassifiedintothreetypesonthebasisofsubunitcomposition,cleavageposition,sequence-specificityandcofactor-requirements.However,aminoacidsequencinghasuncoveredextraordinaryvarietyamongrestrictionenzymesandrevealedthatatthemolecularleveltherearemanymorethanthreedifferentkinds.wTypeIenzymesarecomplex,multisubunit,combinationrestriction-and-modificationenzymesthatcutDNAatrandomfarfromtheirrecognitionsequences.Originallythoughttoberare,wenowknowfromtheanalysisofsequencedgenomesthattheyarecommon.TypeIenzymesareofconsiderablebiochemicalinterestbuttheyhavelittlepracticalvaluesincetheydonotproducediscreterestrictionfragmentsordistinctgel-bandingpatterns.wTypeIIenzymescutDNAatdefinedpositionsclosetoorwithintheirrecognitionsequences.Theyproducediscreterestrictionfragmentsanddistinctgelbandingpatterns,andtheyaretheonlyclassusedinthelaboratoryforDNAanalysisandgenecloning.Ratherthenformingasinglefamilyofrelatedproteins,typeIIenzymesareacollectionofunrelatedproteinsofmanydifferentsorts.TypeIIenzymesfrequentlydiffersoutterlyinaminoacidsequencefromoneanother,andindeedfromeveryotherknownprotein,thattheylikelyaroseindependentlyinthecourseofevolutionratherthandivergingfromcommonancestors.wThemostcommontypeIIenzymesarethoselikeHhaI,HindIIIandNotIthatcleaveDNAwithintheirrecognitionsequences.wEnzymesofthiskindaretheprincipleonesavailablecommercially.MostrecognizeDNAsequencesthataresymmetricbecausetheybindtoDNAashomodimers,butafew,(e.g.,BbvCI:CCTCAGC)recognizeasymmetricDNAsequencesbecausetheybindasheterodimers.Someenzymesrecognizecontinuoussequences(e.g.,EcoRI:GAATTC)inwhichthetwohalf-sitesoftherecognitionsequenceareadjacent,whileothersrecognizediscontinuoussequences(e.g.,BglI:GCCNNNNNGGC)inwhichthehalf-sitesareseparated.Cleavageleavesa3-hydroxylononesideofeachcutanda5-phosphateontheother.wTheyrequireonlymagnesiumforactivityandthecorrespondingmodificationenzymesrequireonlyS-adenosylmethionine.Theytendtobesmall,withsubunitsinthe200350aminoacidrange.wThenextmostcommontypeIIenzymes,usuallyreferredtoastypeIIsarethoselikeFokIandAlwIthatcleaveoutsideoftheirrecognitionsequencetooneside.Theseenzymesareintermediateinsize,400650aminoacidsinlength,andtheyrecognizesequencesthatarecontinuousandasymmetric.Theycomprisetwodistinctdomains,oneforDNAbinding,theotherforDNAcleavage.TheyarethoughttobindtoDNAasmonomersforthemostpart,buttocleaveDNAcooperatively,throughdimerizationofthecleavagedomainsofadjacentenzymemolecules.Forthisreason,sometypeIIsenzymesaremuchmoreactiveonDNAmoleculesthatcontainmultiplerecognitionsites.wThethirdmajorkindoftypeIIenzyme,moreproperlyreferredtoastypeIVarelarge,combinationrestriction-and-modificationenzymes,8501250aminoacidsinlength,inwhichthetwoenzymaticactivitiesresideinthesameproteinchain.Theseenzymescleaveoutsideoftheirrecognitionsequences;thosethatrecognizecontinuoussequences(e.g.,Eco57I:CTGAAG)cleaveonjustoneside;thosethatrecognizediscontinuoussequences(e.g.,BcgI:CGANNNNNNTGC)cleaveonbothsidesreleasingasmallfragmentcontainingtherecognitionsequence.Theaminoacidsequencesoftheseenzymesarevariedbuttheirorganizationareconsistent.TheycompriseanN-terminalDNA-cleavagedomainjoinedtoaDNA-modificationdomainandoneortwoDNAsequence-specificitydomainsformingtheC-terminus,orpresentasaseparatesubunit.Whentheseenzymesbindtotheirsubstrates,theyswitchintoeitherrestrictionmodetocleavetheDNA,ormodificationmodetomethylateit.TypeIIIenzymeswTypeIIIenzymesarealsolargecombinationrestriction-and-modificationenzymes.TheycleaveoutsideoftheirrecognitionsequencesandrequiretwosuchsequencesinoppositeorientationswithinthesameDNAmoleculetoaccomplishcleavage;theyrarelygivecompletedigests.Nolaboratoryuseshavebeendevisedforthem,andnoneareavailablecommercially.一实验目的及背景一实验目的及背景w核酸限制性内切酶是一类能识别双链中特定碱基顺序的核酸水解酶，这些酶都是从原核生物中发现，它们的功能犹似高等功物的免疫系统，用于抗击外来的侵袭。w限制性内切酶以内切方式水解核酸链中的磷酸二酯键，产生的片段端为，端为。限制酶的类型w根据限制酶的识别切割特性，催化条件及是否具有修饰酶活性可分为、型三大类。w类和类限制性内切酶，在同一蛋白分子中兼有甲基化作用及依赖ATP的限制性内切酶活性。w类限制性内切酶结合于特定识别位点，且没有特定的切割位点，酶对其识别位点进行随机切割，很难形成稳定的特异性切割末端。w类限制性内切酶在识别位点上切割，然后从底物上解离下来。w故类和类酶在基因工程中基本不用。型酶w型酶就是通常指的限制性内切酶.w它们能识别双链的特异顺序，并在这个顺序内进行切割，产生特异的片段；w型酶分子量较小，仅需Mg2+作为催化反应的辅助因子，识别顺序一般为个碱基对的反转重复顺序；w型内切酶切割双链产生种不同的切口端突出；端突出和平末端。w正是得益于限制性的内切酶的发现和应用，才使得人们能在体外有目的地对遗传物质进行改造，从而极大地推动了分子生物学的兴旺和发展。酶切反应中应注意以下几个问题：内切酶：w不应混有其它杂蛋白特别是其它内切酶或外切酶的污染；w注意内切酶的识别位点及形成的粘性末端；w内切酶的用量根据内切酶单位和用量而定，通常1u指在适当条件下，1小时内完全酶解ug特定底物所需要的限制性内切酶量，使用中一般以ugDNA对u酶短时间为宜。w同时内切酶体积不能超过反应体系，因内切酶中含甘油，体系中甘油超过会抑制内切酶活力；w内切酶操作应在低温下进行（冰上）；使用时防止操作中对内切酶的污染。：w作为内切酶底物，应该具备一定的纯度，其溶液中不能含酚、氯仿、乙醚、SDS、EDTA、高盐浓度、酒精等，这些因素的存在均不同程度影响限制性内切酶的活力。w这种抑制可通过：w增加酶作用单位数（1020U/ugDNA）、w增大反应体积以稀释可能的抑制剂w或延长反应时间加以克服。反应缓冲液：w反应缓冲液主要由TrisHCl、NaCl、Mg2+组成，其中Mg2+为内切酶辅基；wTrisHCl维持反应体系pH值在之间；wNaCl浓度不同形成种级别的离子强度：w低盐（10mMNaCl)w中盐（50mMNaCl）w高盐（100mMNaCl）w不同的内切酶选择特定的反应缓冲液。酶解温度与时间：w大多数限制酶反应温度为，如EcoR,Hind,BamH,Pst等，也有如Bcl需在下进行反应，w反应时间根据酶的单位与用量之比来定，原则是酶：=：w小时即可，充分酶解。二、实验试剂二、实验试剂w限制性内切酶w（和质粒）wbuffer:50mMTrisHClpH7.5w100mMNaClw10mMMgCl2w无菌水三实验方法三实验方法w按下表分别加入各试剂（注意限制性内切酶最后加入且在冰上操作）于Eppendorf管中。wDNAgw10buffer2.5lw无菌水w内切酶2w总体积：25lw将反应体系充分混匀，并于台式离心机上短暂离心。wEppendorf管封上封口膜于水管中反应小时。w反应结束后加入EDTA至终浓度10mM终止反应。w取l反应液加lLoadingbuffer混匀于琼脂糖凝胶上伏电泳小时。w紫外透射仪上检查实验结果。四结果与分析四结果与分析w记录紫外透射仪上观察到的结果。w分析实验结果的成因。w问题与讨论：w在整个酶切反应过程中应注意哪些问题？w如何选择和限制性内切酶的用量？w反应体系中为何内切酶用量不能超过整个反应体系的？