DNA ligase are used known as molecular glues. There are 3 types of ligases used 1) DNA ligase 2) T4 infected Ecoli DNA ligase 3) terminal deoxynucleotidyl transferase

1. Practical#11
28-12-2018
Ligationof different DNA fragments
The gene of interest is cut from the donor gene and
pasted to the carrying vector. Thus formed recombinant
DNA is transferred into the host organism which has to be
modified genetically. The process of Genetic engineering
is made effective by the action of two enzymes namely,
Restriction enzymes and ligases. Restriction enzymes,
called molecularscissorsare precisely cut thedesired DNA
and its carrying vector, at specific sites , whereas the ligase
enzyme, called Molecular glue are pasted these DNA
fragments into the carrying vector. The process of ligase
enzyme for joining the two ends of DNA strands is called
ligation. In this process, there occurs a synthesis of
phosphodiester bond between the 3’hydroxyl of one
nucleotideand 5’phosphateof another.There are currently
threemethodsfor joining DNA fragments in vitro. The first
of these is DNA ligase(requires NAD with NMN as
leaving group) that covalently joins the annealed
cohesive ends produced by certain restriction enzymes.
The second depends upon the ability of DNA ligase from
phage T4-infected E. coli to catalyse the formation of
phosphodiester bonds between sticky or blunt-ended
fragments. The third utilizes the enzyme terminal

2. deoxynucleotidyl transferase to synthesize
homopolymeric 3′ single-stranded tails at the ends of
fragments. The most commonly used is the T4 DNA ligase
method.
Principle:
In this the 3’-5’ are connected with the help of ligase
enzyme. DNA fragments with either blunt or sticky ends
can be inserted into vector DNA with the help of DNA
ligases. During this purified DNA ligases are given which
covalently join the ends of a restriction fragments and
vector DNA that have complementary ends. To have
complementary ends the vector and the DNA of insert
should be cut With the same Restriction Enzyme.
T4 Ligases:
BacteriophageT4 DNA ligase is a single polypeptidewith
a M.W of 68,000 Dalton requiring ATP as energy
source. The maximal activity pH range is 7.5-8.0. The
enzyme exhibits 40% of its activityat pH 6.9 and 65% at
pH 8.3. The presence of Mg++ ion is required and the
optimal concentration is 10mM. T4 DNA ligase has the
unique ability to join sticky and blunt ended fragments.
Cohesive end ligation is carried out at 12°C to 16°C.
Requirement:

3. Vector DNA,Insert DNA ,Ligase buffer, T4 DNA ligase,
H2O to make total volume 10μl.
Procedure:
1.Take oneclean fresh microfuge tube(Sample)fromthe
rack.
2.In this microfuge tube(Vial), add 5.5µL water, 1.5µL
Vector, 1.5µL insert and 1µL T4 DNA ligase buffer.
3.0.5 µL of T4 DNA Ligase enzyme was added to the
sample tube ( total reaction volume is 10 µl).
4.The vial is kept in the micro centrifuge and just spin
for a few seconds.
5.Incubate the vial at room temperature (22˚C ) or at
16˚C for 2 hours.
6.After 2 hours, the ligated mixture is taken for doing
transformation.
Applications:
 Ligation of cohesive and blunt ends for clonning
 Sealing nicks in Double stranded DNA
 Ligation of synthetic linkers to blunt ends