南开大学基因操作原理第三章.pptVIP

Chapter3CuttingandjoiningDNAmolecular1

?1st?3rd?2ndinvitroinvivo2

PCRRT-PCREndmodification3

OverviewofUsefulEnzymesforMolecularCloning2.RestrictionEndonucleases1)Host-controlledrestrictionandmodification;2)Restrictionenzymeandmodificationenzyme;3)Typesofrestrictionenzyme;4)Nomenclatureofrestrictionenzyme;5)Targetsiteofrestrictionenzyme;6)Isoschizomer;7)Isocaudamer.4

3.DNALigase1)MechanismofDNAligase;2)CharacteristicandapplicationofDNAligase.4.EndModification1)Enzymes;2)Double-linker;3)Adaptors;4)Homopolyertailing.5

61.UsefulEnzymesforMolecularCloning

Theformationofnewcombinationsofheritablematerialbytheinsertionofnucleicacidmoleculeswithsomeenzymes(suchasRestrictionEndonucleases,DNALigase,Polymerase),producedbywhatevermeansoutsidethecell.Enzymeistheformationofbio-engineeringproducts,usuallyfromawildstrainoreukaryotes(suchasyeast)totransformoroptimize.7EnzymeastoolinGenemanipulation

Innature,therearesomeenzymeswithspecificfunctioninmanymicro-organisms.NucleicacidmetabolismDNAreplicationandrepairDegradationofnon-selfDNA8UsefulEnzymeastoolinGenemanipulation

RestrictionendonucleaseMethylaseDNAligaseDNApolymeraseRNApolymeraseKinaseandphosphataseNuclease9EnzymeastoolinGenemanipulation

Host-controlledrestrictionandmodificationRestrictionenzymeandmodificationenzymeTypesofrestrictionenzymesNomenclatureofrestrictionenzymesTargetsiteofrestrictionenzymesIsoschizomerIsocaudamer102.RestrictionEndonuclease

1)Host-controlledRestrictionandModificationPhageinfectsbacteria11

Twoenzymeactivityinhost:RestrictionendonucleaseenzymeModificationmethyltransferase12Restriction-Modefication(R-M)system

E.ColiBE.ColiKModifiedphageλ(B)Modifiedphageλ(K)EOP＝10-4（Restriction）EOP＝10-4（Restriction）EOP=1（Modification）