欢迎来到天天文库
浏览记录
ID:52541696
大小:181.00 KB
页数:19页
时间:2020-04-09
《实验十一 DNA的酶切.ppt》由会员上传分享,免费在线阅读,更多相关内容在PPT专区-天天文库。
1、实验十一DNA的酶切RestrictionEndonucleases: AnOverviewRestrictionenzymeswerediscoveredabout30yearsagoduringinvestigationsintothephenomenonofhost-specificrestrictionandmodificationofbacterialviruses.Bacteriainitiallyresistinfectionsbynewviruses,andthis"restriction"ofviralgrowthstemmedfromendonucleas
2、eswithinthecellsthatdestroyforeignDNAmolecules.Amongthefirstofthese"restrictionenzymes"tobepurifiedwereEcoRIandEcoRIIfromEscherichiacoli,andHindIIandHindIIIfromHaemophilusinfluenzae.TheseenzymeswerefoundtocleaveDNAatspecificsites,generatingdiscrete,gene-sizefragmentsthatcouldbere-joinedinth
3、elaboratory.Researcherswerequicktorecognizethatrestrictionenzymesprovidedthemwitharemarkablenewtoolforinvestigatinggeneorganization,functionandexpression.Astheuseofrestrictionenzymesspreadamongmolecularbiologistsinthelate1970’s,companiessuchasNewEnglandBiolabsbegantosearchformore.Exceptforc
4、ertainviruses,restrictionenzymeswerefoundonlywithinprokaryotes.Manythousandsofbacteriaandarchaehavenowbeenscreenedfortheirpresence.Analysisofsequencedprokaryoticgenomesindicatesthattheyarecommon--allfree-livingbacteriaandarchaeaappeartocodeforthem.Restrictionenzymesareexceedinglyvaried;they
5、rangeinsizefromthediminutivePvuII(157aminoacids)tothegiantCjeI(1250aminoacids)andbeyond.Amongover3,000activitiesthathavebeenpurifiedandcharacterized,morethan250differentsequence-specificitieshavebeendiscovered.Ofthese,over30%werediscoveredandcharacterizedatNewEnglandBiolabs.Thesearchfornews
6、pecificitiescontinues,bothbiochemically,bytheanalysisofcell-extracts,andcomputationally,bytheanalysisofsequencedgenomes.Althoughmostactivitiesencounteredtodayturnouttobeduplicates--isoschizomers--ofexistingspecificities,restrictionenzymeswithnewspecificitiesarefoundwithregularity.Beginningi
7、ntheearly1980’s,NewEnglandBiolabsembarkedonaprogramtocloneandoverexpressthegenesforrestrictionenzymes.Cloningimprovesenzymepuritybyseparatingenzymesfromcontaminatingactivitiespresentinthesamecells.Italsoimprovesenzymeyieldsandgreatlysimplifiespurificatio
此文档下载收益归作者所有