[PDF] DNA Modifying Enzymes

80178_3note_1492433946.pdf B Tech BTE(2015-2019) UNIT I SŁT1205 GNNNTIC NNGINNNRING 1

DNA MODIFYING ENZYMES

Restriction enzymes and DNA ligases represent the cutting and joining functions in MNA manipulaWion. All oWUer enYymeV involveT in geneWic engineering fall unTer WUe broaT caWegory of enYymeV known aV MNA moTifying enYymeV. TUeVe enYymeV are involveT in WUe TegraTaWionH VynWUeViV anT alWeraWion of WUe nucleic aciTV. DNA ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonTV WUaW may occur aW ranTom or aV a conVequence of MNA replicaWion or recombinaWion. In geneWic engineering iW iV uVeT Wo Veal TiVconWinuiWieV in WUe Vugarphosphate chains that arise wUen recombinanW MNA iV maTe by joining MNA moleculeV from TifferenW VourceV. IW can WUerefore be WUougUW of aV molecular glueH wUicU iV uVeT Wo VWick pieceV of MNA WogeWUer.TUiV funcWion iV crucial Wo WUe VucceVV of many experimenWVH anT MNA ligaVe iV WUerefore a key enYyme in geneWic engineering. TUe enYyme uVeT moVW ofWen in experimenWV iV T4 MNA ligaVeH wUicU iV purifieT from N. coli cellV infecWeT wiWU bacWeriopUage T4.AlWUougU WUe enYyme iV moVW efficienW wUen Vealing gapV in fragmenWV WUaW are UelT WogeWUer by coUeVive enTVH iW will alVo join blunW-enTeT MNA moleculeV WogeWUer unTer appropriaWe conTiWionVT4 MNA LigaVe caWalyYeV WUe formaWion of a pUoVpUoTieVWer bonT beWween juxWapoVeT 5G-pUoVpUaWe anT 3G-UyTroxyl Wermini in Tuplex MNA or RNA. TUe enYyme repairV Vingle-VWranT nickV in Tuplex MNAH RNAH or MNAIRNA UybriTV. IW alVo joinV MNA fragmenWV wiWU eiWUer coUeVive or blunW WerminiH buW UaV no acWiviWy on Vingle- VWranTeT nucleic aciTV. TUe T4 MNA LigaVe requireV ATP aV a cofacWor.

RESTRICTION ENDONUCLEASES

¾ Also called restriction enzymes

¾

¾ E. coli bacteria have an enzymatic immune system WUaW recogniYeV anT TeVWroyV foreign MNA ¾ 3,000 enzymes have been identified, around 200 have unique properties, many are purifieT anT available commercially B Tech BTE(2015-2019) UNIT I SŁT1205 GNNNTIC NNGINNNRING 2 Named for bacterial genus, species, strain, and type NxampleJ NcoR1 GenuVJNVcUericUia SpecieVJcoli SWrainJR OrTer TiVcovereTJ 1 NnYymeV recogniYe Vpecific 4-8 bp VequenceV wUicU are palinTromic.

Some enYymeV cuW in a VWaggereT faVUion -

Some enYymeV cuW in a TirecW faVUion

B Tech BTE(2015-2019) UNIT I SŁT1205 GNNNTIC NNGINNNRING 3

Uses of restriction enzymes

¾ RFLP analysis (Restriction Fragment Length Polymorphism)

¾ DNA sequencing

¾ DNA storage librarieV

¾ Transformation

¾ Large scale analysis gene cUipV

TYPES OF DNA MODIFYING ENZYMES

Nuclease enzymes degrade nucleic acids by breaking the phosphodiester bond that holds WUe nucleoWiTeV WogeWUer. ReVWricWion enYymeV are gooT exampleV of enTonucleaVeVH wUicU cuW wiWUin a MNA VWranT.A VeconT group of nucleaVeVH wUicU TegraTe MNA from WUe B Tech BTE(2015-2019) UNIT I SŁT1205 GNNNTIC NNGINNNRING 4 termini of the molecule, are known as exonucleases. Apart from restriction enzymes, WUere are four uVeful nucleaVeV WUaW are ofWen uVeT in geneWic engineering. TUeVe are y Bal 31 y exonuclease III (exonucleases), y deoxyribonuclease I (DNase I) y S1-nucleaVe (endonucleases). TUeVe enYymeV Tiffer in WUeir preciVe moTe of acWion anT proviTe WUe geneWic engineer wiWU a varieWy of VWraWegieV for aWWacking MNA. Nuclease Bal 31 is a complex enzyme. IWV primary acWiviWy iV a faVW- which is coupled with a slow-acWing enTonucleaVe. PUen Łal 31 iV preVenW aW a UigU concenWraWion WUeVe acWiviWieV effecWively VUorWen MNA moleculeV from boWU Wermini.

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