LIGATION OF VECTOR & PASSENGER DNA

1. CHRIST COLLEGE
JAGDALPUR
 TOPIC - LIGATION OF
VECTOR &
PASSENGER DNA

2. SYNOPSIS
INTRODUCTION
STICKY OR COHESIVE
END LIGATION METHOD
BLUNT END LIGATION
HOMOPOLYMER TAILING
METHOD
CONCLUSION
REFRENCE

3. INTRODUCTION
 After isolating a foreign
DNA fragments one has to
join it to its cloning vector to
create a recombinant –DNA
.
 The cloning vector is
isolated and then cleaved
using restriction enzyme ,
the vector so cleaved may
be either sticky or blunt
end.
 The cleaved vectors are
enabled to join with isolated
DNA fragments.

4. There are three methods
used for ligation of
Passenger DNA & Vector
STICKY OR COHESIVE END
LIGATION METHOD
BLUNT END LIGATION
METHOD
HOMOPOLYMER TAILING
METHOD

5. STICKY COHESIVE
END LIGATION
METHOD
 This is relatively efficient process
& has been used extremely to
create recombinant DNAs in-
vitro.
 The foreign DNA fragment&
cloning vector have
complementary sequences &
complementary termini of both
join each other by nicks.
 These nicks are sealed by the
action of the enzyme DNA
Ligase resulting in an intact
recombinant DNA double strand.

7. BLUNT-END LIGATION
METHOD
 The joining of foreign DNA
fragment & its cloning vector,
which are blunt-ended, relies
upon the ability of T4 DNA
ligase.
 The linkers are first ligated to
both ends blunt ended foreign
DNA fragment with the help of T4
DNA ligase & then treated with a
restriction enzyme to produce
sticky ends in them thus
converting blunt ended DNA
fragment into sticky ended one.

8. BLUNT END
LIGATION
 Later are now joined with
appropriate DNA Ligase into
cloning vector that bears
complementary sticky ends
as a result of the action of
same restriction enzyme.
 This produces a recombinant
–DNA.
 Such a blunt end ligation
procedure in which linker as
well as cloning vector are
treated with Eco RI restriction
enzyme to generate sticky
ends complementary to each
other.

10. HOMOPOLYMER
TAILING METHOD
 Homo polymer sequences
can be synthesised by the
enzyme terminal transferase
which is obtained from calf
thymus.
 In this method the
complementary homo
polymer sequence are added
to the ends of DNA molecules
of interest.
 For instance , by adding oligo
(dA) sequences to the 3’end
of one DNA molecule
population & oligo (dT)
sequences to the 3’ ends of
another, the two type of DNA
molecules can be enabled to
join each other resulting in

13. CONCLUSION
 In molecular biology, ligation is the
joining of two nucleic acid fragments
through the action of an enzyme .
 It is essential laboratory procedure in
the molecular cloning of DNA where
by DNA fragments are joined
together to create recombinant DNA
molecules , such as when a foreign
DNA fragments are inserted into a
plasmid .
 The ends of DNA fragments are joined
together by formation of phospho
diester bond between the 3’-Hydroxyl
of one DNA terminus with the 5’-
Phosphoryl of another.

14. REFRENCE
 GENETIC ENGINEERING
OF BIOTECHNOLOGY
 INTERNET

15. THANK YOU