Strategies for cloning PCR products: TA cloning, Topo cloning.pdf

TA cloning, Topo cloning, Ligation independent cloning

1. Strategies for cloning PCR products
Dr. Manikandan Kathirvel M.Sc., Ph.D.,
(NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Reaccredited with "A++" Grade by NAAC
K. Narayanapura, Kothanur(PO)
Bengaluru 560077
Email:[email protected]
ORCID ID: 0000000270066334

2. TA CLONING
TAcloning(alsoknownasrapidcloningorTcloning)isacloningtechniquethatavoidstheuseofrestriction
enzymesandiseasierandquickerthantraditionalcloning.
Thetechniquereliesontheabilityofadenine(A)andthymine(T)(complementarybasepairs)ondifferentDNA
fragmentstohybridizeand,inthepresenceofligase,becomeligatedtogether.

3. TACloningFunctioning
TAcloningisbroughtaboutbytheterminaltransferaseactivityofcertaintypeofDNApolymerasesuchas
theTaqpolymerase.
PCRproductsareusuallyamplifiedusingTaqDNApolymerasewhichpreferentiallyaddsanadeninetothe3'endof
theproduct.Thisenzymeaddsasingle,3'-AoverhangtoeachendofthePCRproduct.
Asaresult,thePCRproductcanbedirectlyclonedintoalinearizedcloningvectorthathavesinglebase
3'-Toverhangsoneachend.SuchvectorsarecalledT-vectors.
ThePCRproductwithAoverhang,ismixedwiththisvectorinhighproportion.Thecomplementary
overhangsofa"T"vectorandthePCRproductwithdAoverhanghybridize.Theresultisarecombinant
DNA,therecombinationbeingbroughtaboutbyDNAligase.

4. Procedure
Diagram of TA Cloning
Creatingtheinsert
TheinsertiscreatedbyPCRusingTaqpolymerase.This
polymeraselacks3'to5'proofreadingactivityand,withahigh
probability,addsasingle,3'-adenineoverhangtoeachendof
thePCRproduct.ItisbestifthePCRprimershaveguaninesat
the5'endasthismaximizesprobabilityofTaqDNA
polymeraseaddingtheterminaladenosineoverhang.
Thermostablepolymerasescontainingextensive3´to5´
exonucleaseactivityshouldnotbeusedastheydonotleavethe
3´adenine-overhangs.
Creatingthevector
Thetargetvectorislinearizedandcutwithablunt-end
restrictionenzyme.Thisvectoristhentailedwith
dideoxythymidinetriphosphate(ddTTP)usingterminal
transferase.ItisimportanttouseddTTPtoensuretheaddition
ofonlyoneTresidue.Thistailingleavesthevectorwitha
single3'-overhangingthymineresidueoneachbluntend.

5. Examples
pLUG®andpLUG®-MultiTA-cloningvectorswere
designedfortheconvenientanddirectPCRcloningand
servingacredibleblue/whitecolonyselection.
FeaturesofthepLUGTA-cloningVectors
Multiplecloningregion
ThemultiplecloningregionislocatedaroundtheTA-cloningsiteinthepLUG
TA-cloningvectors.Therestriction-enzymerecognitionsitesofpLUGcloning
vectoraroundtheTA-cloningsitearelocatedinmirror-repeatpattern.
Originofreplication
ThepLUGTA-cloningvectorhasf1andColE1originsofreplicationwhichis
responsibleforthereplicationofplasmid.
Ampicillinresistancegene
ThepLUGTA-cloningvectorshaveblageneencodingforbeta-lactamasethat
conferresistancetoampicillin.
LacZalphasequence
ThefragmentoflacZalphasequenceinthepLUGTA-cloningvectorsisableto
complementbetagalactosidaseactivity.ThelacZalphasequencereducesthetime
toscreenforpositiveclones.
M13forward/reverseprimingsites
TheM13forward/reverseprimingsitesinthepLUGTA-cloningvectorsallow
convenientsequencing.

6. TOPO TA cloning ofTaq-amplified DNA

7. TOPO –TA cloning
TheTATOPOcloningtechniquereliesontheabilityofadenine(A)and
thymine(T)(complementarybasepairs)ondifferentDNAfragments
tohybridizeand,inthepresenceofligaseortopoisomerase,become
ligatedtogether.
TheinsertiscreatedbyPCRusingTaqDNApolymerase,apolymerase
thatlacks3'to5'proofreadingactivityandwithahighprobabilityaddsa
single,3'-adenineoverhangtoeachendofthePCRproduct.
CloningfragmentsamplifiedbyeitherTaqorPfupolymeraseasTaq
polymerase(unlikePfu)leavesanextra"A"nucleotideatthe3'end
duringamplification.
ThekeytoTOPOcloningistheenzymeDNAtopoisomeraseI,which
functionsbothasarestrictionenzymeandasaligase.
ItsbiologicalroleistocleaveandrejoinDNAduringreplication.
VacciniavirustopoisomeraseIspecificallyrecognizesthepentameric
sequence5´-(C/T)CCTT-3´andformsacovalentbondwiththephosphate
groupattachedtothe3´thymidine.
ItcleavesoneDNAstrand,enablingtheDNAtounwind.Theenzyme
thenreligatestheendsofthecleavedstrandandreleasesitselffromthe
DNA.
Toharnessthereligatingactivityoftopoisomerase,TOPOvectorsare
providedlinearizedwithtopoisomeraseIcovalentlyboundtoeach3´
phosphate.
ThisenablesthevectorstoreadilyligateDNAsequenceswithcompatible
ends(Figures1,2).
Theligationiscompleteinonly5minutesatroomtemperature.
Figure 1. TOPO TA cloning ofTaq-amplified DNA.
"Stickyend"TOPOTAcloning

8. Procedure:
TOPOvectorsaredesignedinsuchawaythattheycarry
thisspecificsequence5´-(C/T)CCTT-3'atthetwolinear
ends.
ThelinearvectorDNAalreadyhasthetopoisomerase
enzymecovalentlyattachedtobothofitsstrands'free3'
ends.ThisisthenmixedwithPCRproducts.
Whenthefree5'endsofthePCRproductstrandsattachto
thetopoisomerase/3'endofeachvectorstrand,thestrands
arecovalentlylinkedbythealreadyboundtopoisomerase.
Thisreactionproceedsefficientlywhenthissolutionis
incubatedatroomtemperaturewithrequiredsalt.

9. Ligation Independent Cloning (LIC)
LIC is a cloning method that makes use of annealing of single-stranded complementary overhangs on the target vector and
a PCR-generated insert of at least 12 bases overlap region.
Single-stranded overhangs can be generated by using T4 DNA polymerase and only one dNTPin the reaction mix, leading
to an equilibrium of 3'->5'-exonuclease and 5'->3'-polymerase activity at the site of the first occurrence of this nucleotide.
The incubation is done with T4 DNA pol. for 30 min. and stopped by adding dCTPto the reaction mix.
After annealing of vector and insert, the mixture is used to transformE. coli.