Recombinant DNA -based molecular techniques (part I) DNA ...
Transcript of Recombinant DNA -based molecular techniques (part I) DNA ...
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RecombinantDNA-basedmoleculartechniques(partI)DNAcloningProf.MamounAhramSecondsemester2021
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WhatisDNAcloning?
DNAcloningisatechniquethatallowsforamplifyingaDNAsegmentintomany,manycopiesinabiologicalsystem.Itusuallyinvolves:
TheformationofarecombinantDNAcomposedofavector(acarrier;usuallyabacterialplasmid)andprotein-codinggene usingrestrictionendonucleases.Insertionintobacteria.
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Restrictionendonucleases
EndonucleassareezymesthatdegradeDNAwithinthemolecule.Restrictionendonucleases:Bacterialenzymesthatrecognizeandcut(break)thephosphodiesterbondbetweennucleotidesatspecific sequences(4- to8-bprestrictionsites)generatingrestrictionfragments.
Restriction endonuclease
Restriction site
Restriction fragments
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Palindromicsequences
Thesequencesrecognizedbyrestrictionendonucleases—theirsitesofaction—readthesamefromlefttorightastheydofromrighttoleft(onthecomplementarystrand).
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Typesofcutsbyrestrictionendonucleases
RestrictionenzymescutDNAintwodifferentways:Blunt:enzymescutatthesamepositiononbothstrands givingabluntendedfragments.Staggered(off-center):enzymescutthetwoDNAstrandsatdifferentpositionsgeneratingstickyorcohesiveends.
TheDNAfragmentshaveshortsingle-strandedoverhangsateachend.
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Zoomintothestickyends
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DNAligase
ItcovalentlyjoinsDNAends(example,restrictionfragments)bycatalyzingtheATP-dependentformationofphosphodiesterbondsbetweenthe3’-hydroxylgroupofonestrandandthe5’-phosphateendofanotherstrand.
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Cloning
Cloningmeansthatyoumakeseveralcopiesofonething.Acloneisageneticallyidenticalpopulation,whetheroforganisms,cells,viruses,orDNAmolecules.Everymemberofthepopulationisderivedfromasinglecell,virus,orDNAmolecule.
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HowdowecloneaDNAmolecule?
aDNAfragmentofinterestisinsertedintoaDNAcarrier (calledavector)thatcanbereplicated.TheresultingDNAmoleculeiswhatisknownasarecombinantDNAmolecule.
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Usingplasmidsasvectors
Bacterialplasmids areconsideredexcellentvectors.ThesearebacterialcircularDNAthatisnotpartofthemaincircularDNAchromosomeofthebacterium.Aplasmidexistsasaclosedcircleandreplicatesindependentlyofthemainbacterialgenome.
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Featuresofplasmids
Mostplasmidvectorscontainatleastthreeessential partsrequiredforDNAcloning:
Theycanreplicate.Theycanbeselectedfor/againstbyaninternaldrug-resistancegene(selectablemarker).AforeignDNAfragmentcanbeinserted(aninsert)intoit.
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MakingofrecombinantDNA
BothDNAfragments(theDNAtobeclonedandavector)arecutbythesamerestrictionendonucleasethatmakesDNAfragmentswithsamesticky-endsthathybdridizetoeachother,whenmixed.
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Cell clones
Cloned DNA
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Overviewofgeneexpression
Shine-DalgarnoSequence
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Expressionvectors Expressionvectorscontainadditionalsequences:
PromotersequencesupstreamofgenetobeinsertedRibosomalbindingsequences(Shine-Dalgarno[SD]sequences)Transcriptionterminationsequence
Theproteinisexpressedandpurified.Examples:insulin,growthhormone,plasminogenactivator,erythropoietin
MWmarker
Novector
+vector
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Howdowedealwithintronsinhumangenes?
Thechallenge Thesolution
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Challenges
InternaldisulfidebondsinbacteriaNopost-translationalmodification(example:glycosylation)MisfoldingDegradation
Solution:useaeukaryoticsystemsuchasyeast
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Proteintaggingorcreationofproteinhybrids
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Proteinscanbe"tagged"
Aprotein-encodinggeneisclonedinaspecialvectorcontainingataggeneproducingaproteinwithanextrasequenceofaminoacidscalledtags.Thesetagsalloweasyproteinpurificationanddetection.
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Usesofproteintags
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MajorproteinandepitopetagsName Aminoacids Detection Purification
FLAG DYKDDDDK antibody FLAGpeptide
Greenfluorescentproteins(GFP)
~220aaprotein antibodyorfluorescence None
Glutathione Stransferase(GST) 218aaprotein antibody glutathione
HA YPYDVPDYA antibody HApeptide
Poly-His HHHHHH antibody nickel,imidazole
Myc EQKLISEED antibody Myc peptide
V5 GKPIPNPLLGLDST antibody V5peptide
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Histag
Theadditionofsixhistidinestoaproteinwouldallowforpurificationusingbreadswithboundnickelions.
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PurificationofGST-taggedproteins
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ProductionofarecombinantproteinGeneA GeneB
Recombinantgene
ProteinA ProteinB
Recombinantprotein
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ProductionofarecombinantproteinGeneA GeneB
Recombinantgene
ProteinA ProteinB
Recombinantprotein
Domain Domain Domain Domain
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GFP-taggedproteinsThepowerofdomains
GreenFluorescentProtein(GFP)allowsforproteindetectionratherthanforpurificationpurposes.
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Aworldofpossibilities