實驗十一酶切

實驗十一酶切第一頁，共十九頁，2022年，8月28日RestrictionEndonucleases:

AnOverviewRestrictionenzymeswerediscoveredabout30yearsagoduringinvestigationsintothephenomenonofhost-specificrestrictionandmodificationofbacterialviruses.Bacteriainitiallyresistinfectionsbynewviruses,andthis"restriction"ofviralgrowthstemmedfromendonucleaseswithinthecellsthatdestroyforeignDNAmolecules.Amongthefirstofthese"restrictionenzymes"tobepurifiedwereEcoR

IandEcoRIIfromEscherichiacoli,andHindIIandHindIIIfromHaemophilusinfluenzae.TheseenzymeswerefoundtocleaveDNAatspecificsites,generatingdiscrete,gene-sizefragmentsthatcouldbere-joinedinthelaboratory.Researcherswerequicktorecognizethatrestrictionenzymesprovidedthemwitharemarkablenewtoolforinvestigatinggeneorganization,functionandexpression.Astheuseofrestrictionenzymesspreadamongmolecularbiologistsinthelate1970’s,companiessuchasNewEnglandBiolabsbegantosearchformore.Exceptforcertainviruses,restrictionenzymeswerefoundonlywithinprokaryotes.Manythousandsofbacteriaandarchaehavenowbeenscreenedfortheirpresence.Analysisofsequencedprokaryoticgenomesindicatesthattheyarecommon--allfree-livingbacteriaandarchaeaappeartocodeforthem.Restrictionenzymesareexceedinglyvaried;theyrangeinsizefromthediminutivePvuII(157aminoacids)tothegiantCjeI(1250aminoacids)andbeyond.Amongover3,000activitiesthathavebeenpurifiedandcharacterized,morethan250differentsequence-specificitieshavebeendiscovered.Ofthese,over30%werediscoveredandcharacterizedatNewEnglandBiolabs.第二頁，共十九頁，2022年，8月28日Thesearchfornewspecificitiescontinues,bothbiochemically,bytheanalysisofcell-extracts,andcomputationally,bytheanalysisofsequencedgenomes.Althoughmostactivitiesencounteredtodayturnouttobeduplicates--isoschizomers--ofexistingspecificities,restrictionenzymeswithnewspecificitiesarefoundwithregularity.Beginningintheearly1980’s,NewEnglandBiolabsembarkedonaprogramtocloneandoverexpressthegenesforrestrictionenzymes.Cloningimprovesenzymepuritybyseparatingenzymesfromcontaminatingactivitiespresentinthesamecells.Italsoimprovesenzymeyieldsandgreatlysimplifiespurification,anditprovidesthegenesforsequencingandanalysis,andtheproteinsforx-raycrystallography.Restrictionenzymesprotectbacteriafrominfectionsbyviruses,anditisgenerallyacceptedthatthisistheirroleinnature.Theyfunctionasmicrobialimmunesystems.WhenastrainofE.colilackingarestrictionenzymeisinfectedwithavirus,mostvirusparticlescaninitiateasuccessfulinfection.Whenthesamestraincontainsarestrictionenzyme,however,theprobabilityofsuccessfulinfectionplummets.Thepresenceofadditionalenzymeshasamultiplicativeeffect;acellwithfourorfiveindependentrestrictionenzymescouldbevirtuallyimpregnable.第三頁，共十九頁，2022年，8月28日

Restrictionenzymesusuallyoccurincombinationwithoneortwomodificationenzymes(DNA-methyltransferases)thatprotectthecell’sownDNAfromcleavagebytherestrictionenzyme.ModificationenzymesrecognizethesameDNAsequenceastherestrictionenzymethattheyaccompany,butinsteadofcleavingthesequence,theymethylateoneofthebasesineachoftheDNAstrands.ThemethylgroupsprotrudeintothemajorgrooveofDNAatthebindingsiteandpreventtherestrictionenzymefromactinguponit.Together,arestrictionenzymeandits"cognate"modificationenzyme(s)formarestriction-modification(R-M)system.InsomeR-Msystemstherestrictionenzymeandthemodificationenzyme(s)areseparateproteinsthatactindependentlyofeachother.Inothersystems,thetwoactivitiesoccurasseparatesubunits,orasseparatedomains,ofalarger,combined,restriction-and-modificationenzyme.第四頁，共十九頁，2022年，8月28日Restrictionenzymesaretraditionallyclassifiedintothreetypesonthebasisofsubunitcomposition,cleavageposition,sequence-specificityandcofactor-requirements.However,aminoacidsequencinghasuncoveredextraordinaryvarietyamongrestrictionenzymesandrevealedthatatthemolecularleveltherearemanymorethanthreedifferentkinds.TypeIenzymesarecomplex,multisubunit,combinationrestriction-and-modificationenzymesthatcutDNAatrandomfarfromtheirrecognitionsequences.Originallythoughttoberare,wenowknowfromtheanalysisofsequencedgenomesthattheyarecommon.TypeIenzymesareofconsiderablebiochemicalinterestbuttheyhavelittlepracticalvaluesincetheydonotproducediscreterestrictionfragmentsordistinctgel-bandingpatterns.第五頁，共十九頁，2022年，8月28日TypeIIenzymescutDNAatdefinedpositionsclosetoorwithintheirrecognitionsequences.Theyproducediscreterestrictionfragmentsanddistinctgelbandingpatterns,andtheyaretheonlyclassusedinthelaboratoryforDNAanalysisandgenecloning.Ratherthenformingasinglefamilyofrelatedproteins,type

IIenzymesareacollectionofunrelatedproteinsofmanydifferentsorts.TypeIIenzymesfrequentlydiffersoutterlyinaminoacidsequencefromoneanother,andindeedfromeveryotherknownprotein,thattheylikelyaroseindependentlyinthecourseofevolutionratherthandivergingfromcommonancestors.ThemostcommontypeIIenzymesarethoselikeHha

I,HindIIIandNotIthatcleaveDNAwithintheirrecognitionsequences.Enzymesofthiskindaretheprincipleonesavailablecommercially.MostrecognizeDNAsequencesthataresymmetricbecausetheybindtoDNAashomodimers,butafew,(e.g.,BbvCI:CCTCAGC)recognizeasymmetricDNAsequencesbecausetheybindasheterodimers.Someenzymesrecognizecontinuoussequences(e.g.,

EcoR

I:GAATTC)inwhichthetwohalf-sitesoftherecognitionsequenceareadjacent,whileothersrecognizediscontinuoussequences(e.g.,BglI:GCCNNNNNGGC)inwhichthehalf-sitesareseparated.Cleavageleavesa3′-hydroxylononesideofeachcutanda5′-phosphateontheother.TheyrequireonlymagnesiumforactivityandthecorrespondingmodificationenzymesrequireonlyS-adenosylmethionine.Theytendtobesmall,withsubunitsinthe200–350aminoacidrange.第六頁，共十九頁，2022年，8月28日ThenextmostcommontypeIIenzymes,usuallyreferredtoas‘typeIIs"arethoselikeFokIandAlwIthatcleaveoutsideoftheirrecognitionsequencetooneside.

Theseenzymesareintermediateinsize,400–650aminoacidsinlength,andtheyrecognizesequencesthatarecontinuousandasymmetric.Theycomprisetwodistinctdomains,oneforDNAbinding,theotherforDNAcleavage.TheyarethoughttobindtoDNAasmonomersforthemostpart,buttocleaveDNAcooperatively,throughdimerizationofthecleavagedomainsofadjacentenzymemolecules.Forthisreason,sometypeIIsenzymesaremuchmoreactiveonDNAmoleculesthatcontainmultiplerecognitionsites.ThethirdmajorkindoftypeIIenzyme,moreproperlyreferredtoas"typeIV"arelarge,combinationrestriction-and-modificationenzymes,850–1250aminoacidsinlength,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.第七頁，共十九頁，2022年，8月28日TypeIIIenzymesTypeIIIenzymesarealsolargecombinationrestriction-and-modificationenzymes.TheycleaveoutsideoftheirrecognitionsequencesandrequiretwosuchsequencesinoppositeorientationswithinthesameDNAmoleculetoaccomplishcleavage;theyrarelygivecompletedigests.Nolaboratoryuseshavebeendevisedforthem,andnoneareavailablecommercially.

第八頁，共十九頁，2022年，8月28日一．實驗?zāi)康募氨尘?/p>

核酸限制性內(nèi)切酶是一類能識別雙鏈ＤＮＡ中特定堿基順序的核酸水解酶，這些酶都是從原核生物中發(fā)現(xiàn)，它們的功能猶似高等功物的免疫系統(tǒng)，用于抗擊外來ＤＮＡ的侵襲。限制性內(nèi)切酶以內(nèi)切方式水解核酸鏈中的磷酸二酯鍵，產(chǎn)生的ＤＮＡ片段５’端為Ｐ，３’端為ＯＨ。第九頁，共十九頁，2022年，8月28日限制酶的類型根據(jù)限制酶的識別切割特性，催化條件及是否具有修飾酶活性可分為Ⅰ、Ⅱ、Ⅲ型三大類。Ⅰ類和Ⅲ類限制性內(nèi)切酶，在同一蛋白分子中兼有甲基化作用及依賴ATP的限制性內(nèi)切酶活性。Ⅰ類限制性內(nèi)切酶結(jié)合于特定識別位點，且沒有特定的切割位點，酶對其識別位點進行隨機切割，很難形成穩(wěn)定的特異性切割末端。Ⅲ類限制性內(nèi)切酶在識別位點上切割，然后從底物上解離下來。故Ⅰ類和Ⅲ類酶在基因工程中基本不用。第十頁，共十九頁，2022年，8月28日Ⅱ型酶Ⅱ型酶就是通常指的ＤＮＡ限制性內(nèi)切酶.它們能識別雙鏈ＤＮＡ的特異順序，并在這個順序內(nèi)進行切割，產(chǎn)生特異的ＤＮＡ片段；Ⅱ型酶分子量較小，僅需Mg2+作為催化反應(yīng)的輔助因子，識別順序一般為４～６個堿基對的反轉(zhuǎn)重復(fù)順序；Ⅱ型內(nèi)切酶切割雙鏈ＤＮＡ產(chǎn)生３種不同的切口－－５’端突出；３’端突出和平末端。正是得益于限制性的內(nèi)切酶的發(fā)現(xiàn)和應(yīng)用，才使得人們能在體外有目的地對遺傳物質(zhì)ＤＮＡ進行改造，從而極大地推動了分子生物學(xué)的興旺和發(fā)展。第十一頁，共十九頁，2022年，8月28日酶切反應(yīng)中應(yīng)注意以下幾個問題：１．內(nèi)切酶：不應(yīng)混有其它雜蛋白特別是其它內(nèi)切酶或外切酶的污染；注意內(nèi)切酶的識別位點及形成的粘性末端；內(nèi)切酶的用量根據(jù)內(nèi)切酶單位和ＤＮＡ用量而定，通常1u指在適當(dāng)條件下，1小時內(nèi)完全酶解１ug特定ＤＮＡ底物所需要的限制性內(nèi)切酶量，使用中一般以１ugDNA對２－３u酶短時間為宜。同時內(nèi)切酶體積不能超過反應(yīng)體系１０％，因內(nèi)切酶中含５０％甘油，體系中甘油超過５％會抑制內(nèi)切酶活力；內(nèi)切酶操作應(yīng)在低溫下進行（冰上）；使用時防止操作中對內(nèi)切酶的污染。第十二頁，共十九頁，2022年，8月28日２．ＤＮＡ：作為內(nèi)切酶底物，ＤＮＡ應(yīng)該具備一定的純度，其溶液中不能含酚、氯仿、乙醚、SDS、EDTA、高