ligation independent cloning

8/7/2019 ligation independent cloning

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E

E

INDEPENDENTINDEPENDENT

CLONINGCLONING

LIGATION

LIGATION

LIGATIONINDEPENDENTCLONING


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FEATURES

Increased speed and efficiency of cloning No dependence on restriction sites Allows direct cloning of PCR product into

a particular site in plasmid vector Eliminates the need to ligate the PCRproducts into vector Avoids problems caused by extendase

activity of thermostable polymerases at 3end of PCR product


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T4 DNA POLYMERASE

.3 5 exonuclease activity .5 3 polymerase activity Use for filling or labeling.reactions Only one dNTP used in the reaction.mixture dNTP should be in high concentration.to overcome exonuclease activity .Act on ssDNA as well as on dsDNA


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, .ectors like pET pBAC.hanging sequences- arget gene containing 12 14 nt complementtriction sites can be incorporated . (Novy

at the vector insert junction occurs wi

LIC VECTORS & INSERTS


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MECHANISM

T4 DNA polymerase removes nucleotidesfrom linearized vector until it encounters complementary residue of.single dNTP in reaction mixture 5 3 polymerase activity of enzyme.counteracts the exonuclease activity Same with the PCR product which hasoverhang at 5 complementary to vectoroverhang sequences .


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CONTD

The gaps or overhangs in the annealed complex of LIC repairedby bacterial repair mechanism

( Chuan e t a l . , 1997)


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STRATEGY


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to generate ssdna tails at 3 end of PCR product & ve

.I Exonucleaseresection


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Strategy


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CONTD

.2 URACIL DNA GLYCOSYLASECLONING


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Strategy


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LIC VECTORS


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VECTORS(CONTD)

-The pET 32 LIC Vector is designed for-cloning and high level expression ofpeptide sequences fused to the 109( ) ( , )amino acid aa 11 675 Da thioredoxin( , )t r x A T r x T a g .protein, -In addition pET 32 LIC encodes the 6 aa His Tag and 15 aa S Tag sequences

upstream of the cloning site forsimple detection and purification oftarget proteins


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Primers


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Primers(contd)

The sense primer encodes the last four amino( )acids of the enterokinase EK cleavage site-plus the C terminal flanking amino acid Met or( ). ( , , )Ile ATX Ile ATC ATA ATT at this positionrecreates the natural EK site found intrypsinogen

The antisense primer may encode an inframe stop-codon or allow read through to the vector-encoded stop codon present after the C terminalHis Tag sequence


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-The pET 32 LIC Vector also carries theT7 ,l ac pr o mo te r T7 transcription,terminator ,l ac I g e n e pBR322 origin of,replication f1 origin for single stranded,plasmid production and bl a gene for( )ampicillin carbenicillin resistance


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PROTEIN PRODUCTION

,For protein production a recombinant plasmidis prepared from NovaBlue and transformedinto expression( ) ( ) ,Host strains BL21 DE3 or BL21 DE3 pLysS .which are lysogenic for bacteriophage lDE3The DE3 strains possess a chromosomal copyof the T7 RNA polymerase geneunder the

control of the .l a c U V 5 p ro mo t er


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( )AD494 DE3 hosts allow the formation ofdisulfide bonds in the .E coli;cytoplasm soluble proteins containingdisulfide bonds may therefore foldproperly in these strains


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ADVANTAGES

,To study DNA repair in bacteria, .yeast mammals -Eliminating time consuming an

occasionally problematic step of.ligation Efficient because only desired.product is formed &Strategy is highly reproducible.efficient


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?QUESTIONS


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Thank you


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LIGATION INDEPENDENT CLONING


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LIGATION INDEPENDENT CLONING


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S tag


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S tag

-The recognized S Tag epitope represents the amino acid sequence KETAAAKFERQHMDSderived from pancreatic ribonuclease A

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ligation independent cloning