AT I O N

L IG

INDEPENDENT

CLONING
EE

LIGA
TION
INDE
PEND
E NT C
LONI
NG
 FEATURES
ü

üIncreased speed and efficiency of cloning

üNo dependence on restriction sites
üAllows direct cloning of PCR product into
a particular site in plasmid vector
üEliminates the need to ligate the PCR
products into vector
üAvoids problems caused by extendase
activity of thermostable polymerases at 3 ’
end of PCR product
 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 .
 LIC VECTORS & INSERTS

vectors like pET , pBAC .

hanging sequences .

target gene containing 12 - 14 nt complement

striction sites can be incorporated . ( Novy
n at the vector – insert junction occurs wi
 MECHANISM

•T 4 DNA polymerase removes nucleotides

from 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 has
overhang at 5 ‘ complementary to vector
overhang sequences .
 CONTD……

•The gaps or overhangs in the

annealed complex of LIC repaired
by bacterial repair mechanism
•
•
•
•

(Chuan et al ., 1997)
STRATEGY
s to generate ssdna tails at 3’ end of PCR product & vec

I. Exonuclease
resection
Strategy
 CONTD……

2 . URACIL DNA GLYCOSYLASE

CLONING
Strategy
LIC VECTORS
 VECTORS(CONTD)

The pET - 32 LIC Vector is designed for

cloning and high - level expression of
peptide sequences fused to the 109
amino acid ( aa ) ( 11 , 675 Da ) thioredoxin
( trxA , Trx • Tag ™) protein .
In addition , pET - 32 LIC encodes the 6
aa His • Tag® and 15 aa S • Tag ™ sequences
upstream of the cloning site for
simple detection and purification of
target proteins
Primers
 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 position
recreates the natural EK site found in
trypsinogen

The antisense primer may encode an inframe stop

codon or allow read through to the vector -
encoded stop codon present after the C - terminal
His • Tag® sequence
The pET - 32 LIC Vector also carries the
T7 lac promoter , T7 transcription
terminator , lacI gene , pBR322 origin of
replication , f1 origin for single stranded
plasmid production , and bla gene for
ampicillin ( carbenicillin ) resistance
 PROTEIN PRODUCTION

For protein production , a recombinant plasmid

is prepared from NovaBlue and transformed
into expression

Host strains BL21 ( DE3 ) or BL21 ( DE3 ) pLysS ,

which are lysogenic for bacteriophage lDE3 .
The DE3 strains possess a chromosomal copy
of the T7 RNA polymerase geneunder the
control of the lacUV5 promoter .
AD494 ( DE3 ) hosts allow the formation of
disulfide bonds in the E . coli
cytoplasm ; soluble proteins containing
disulfide bonds may therefore fold
properly in these strains
 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 .
?
QUESTIONS
Thank you
LIGATION INDEPENDENT CLONING
 S tag

The recognized S - Tag epitope represents

the amino acid sequence KETAAAKFERQHMDS
derived from pancreatic ribonuclease A