RecombinantDNA-basedmoleculartechniques(partI)DNAcloningProf.MamounAhramSecondsemester2021

WhatisDNAcloning?

DNAcloningisatechniquethatallowsforamplifyingaDNAsegmentintomany,manycopiesinabiologicalsystem.Itusuallyinvolves:

TheformationofarecombinantDNAcomposedofavector(acarrier;usuallyabacterialplasmid)andprotein-codinggene usingrestrictionendonucleases.Insertionintobacteria.

Restrictionendonucleases

EndonucleassareezymesthatdegradeDNAwithinthemolecule.Restrictionendonucleases:Bacterialenzymesthatrecognizeandcut(break)thephosphodiesterbondbetweennucleotidesatspecific sequences(4- to8-bprestrictionsites)generatingrestrictionfragments.

Restriction endonuclease

Restriction site

Restriction fragments

Palindromicsequences

Thesequencesrecognizedbyrestrictionendonucleases—theirsitesofaction—readthesamefromlefttorightastheydofromrighttoleft(onthecomplementarystrand).

Typesofcutsbyrestrictionendonucleases

RestrictionenzymescutDNAintwodifferentways:Blunt:enzymescutatthesamepositiononbothstrands givingabluntendedfragments.Staggered(off-center):enzymescutthetwoDNAstrandsatdifferentpositionsgeneratingstickyorcohesiveends.

TheDNAfragmentshaveshortsingle-strandedoverhangsateachend.

Zoomintothestickyends

DNAligase

ItcovalentlyjoinsDNAends(example,restrictionfragments)bycatalyzingtheATP-dependentformationofphosphodiesterbondsbetweenthe3’-hydroxylgroupofonestrandandthe5’-phosphateendofanotherstrand.

Cloning

Cloningmeansthatyoumakeseveralcopiesofonething.Acloneisageneticallyidenticalpopulation,whetheroforganisms,cells,viruses,orDNAmolecules.Everymemberofthepopulationisderivedfromasinglecell,virus,orDNAmolecule.

HowdowecloneaDNAmolecule?

aDNAfragmentofinterestisinsertedintoaDNAcarrier (calledavector)thatcanbereplicated.TheresultingDNAmoleculeiswhatisknownasarecombinantDNAmolecule.

Usingplasmidsasvectors

Bacterialplasmids areconsideredexcellentvectors.ThesearebacterialcircularDNAthatisnotpartofthemaincircularDNAchromosomeofthebacterium.Aplasmidexistsasaclosedcircleandreplicatesindependentlyofthemainbacterialgenome.

Featuresofplasmids

Mostplasmidvectorscontainatleastthreeessential partsrequiredforDNAcloning:

Theycanreplicate.Theycanbeselectedfor/againstbyaninternaldrug-resistancegene(selectablemarker).AforeignDNAfragmentcanbeinserted(aninsert)intoit.

MakingofrecombinantDNA

BothDNAfragments(theDNAtobeclonedandavector)arecutbythesamerestrictionendonucleasethatmakesDNAfragmentswithsamesticky-endsthathybdridizetoeachother,whenmixed.

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Cell clones

Cloned DNA

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Overviewofgeneexpression

Shine-DalgarnoSequence

Expressionvectors Expressionvectorscontainadditionalsequences:

PromotersequencesupstreamofgenetobeinsertedRibosomalbindingsequences(Shine-Dalgarno[SD]sequences)Transcriptionterminationsequence

Theproteinisexpressedandpurified.Examples:insulin,growthhormone,plasminogenactivator,erythropoietin

MWmarker

Novector

+vector

Howdowedealwithintronsinhumangenes?

Thechallenge Thesolution

Challenges

InternaldisulfidebondsinbacteriaNopost-translationalmodification(example:glycosylation)MisfoldingDegradation

Solution:useaeukaryoticsystemsuchasyeast

Proteintaggingorcreationofproteinhybrids

Proteinscanbe"tagged"

Aprotein-encodinggeneisclonedinaspecialvectorcontainingataggeneproducingaproteinwithanextrasequenceofaminoacidscalledtags.Thesetagsalloweasyproteinpurificationanddetection.

Usesofproteintags

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

Histag

Theadditionofsixhistidinestoaproteinwouldallowforpurificationusingbreadswithboundnickelions.

PurificationofGST-taggedproteins

ProductionofarecombinantproteinGeneA GeneB

Recombinantgene

ProteinA ProteinB

Recombinantprotein

ProductionofarecombinantproteinGeneA GeneB

Recombinantgene

ProteinA ProteinB

Recombinantprotein

Domain Domain Domain Domain

GFP-taggedproteinsThepowerofdomains

GreenFluorescentProtein(GFP)allowsforproteindetectionratherthanforpurificationpurposes.

Aworldofpossibilities