Understanding DNA Ligation Techniques for Molecular Biology Applications

 
L
I
G
A
T
I
O
N
 
O
F
 
D
N
A
F
R
A
G
M
E
N
T
S
 
W
I
T
H
 
V
E
C
T
O
R
S
 
L
I
G
A
T
I
O
N
 
O
F
 
D
N
A
 
F
R
A
G
M
E
N
T
 
W
I
T
H
V
E
C
T
O
R
S
 
DNA 
ligation 
is 
the act 
of 
joining together DNA
strands with covalent bonds 
with 
the 
aim 
of
making 
new viable DNA or
 
plasmid.
There are currently three 
methods 
for joining 
DNA
fragments 
in
 
vitro.
 
 
The 
1st 
of these is 
DNA 
ligase that covalently
joins the annealed cohesive ends produced by
certain restriction
 
enzymes.
 
 
The 
2nd depends upon the ability of 
DNA 
ligase
from 
phage T4-infected 
E. 
coli 
to 
catalyse the
formation of phosphodiester
 bonds
.
 
The 
3rd 
utilizes the enzyme 
terminal
deoxynucleotidyl transferase 
to 
synthesize
homopolymeric 3′ single-stranded tails at the
ends of fragments.
E.coli 
and phage 
T4 
encode an enzyme 
DNA
ligase -T4 enzyme requires 
ATP while 
the E.
coli enzyme requires
 
NAD+.
DNA 
fragments with either 
sticky 
ends or blunt
ends 
can 
be inserted into vector 
DNA 
with 
the
aid of 
DNA 
ligases.
 
L
I
N
K
E
R
S
 
&
 
A
D
A
P
T
O
R
S
 
Linker 
is a 
synthetic ,short and 
known 
double
stranded oligonucleotides sequence.
Having blunted ends on both sides and
restriction
 
sites.
E.Coli 
DNA 
ligase 
will 
not catalyse blunt end
ligation except under special
 
condition.
Linker can be ligated 
to 
both ends of the
foreign
 DNA.
 
Treatment with R.E produces 
sticky 
ends after
ligation with target
 
DNA.
Sticky ends are desirable for DNA cloning
experiments.
One 
drawback 
is 
R.E. 
used to 
generate
cohesive 
end 
in 
the linker 
will 
also cut 
foreign
DNA 
at internal 
sites.
Solution to the problem is 
to choose 
another
restriction enzyme or 
to 
methylate internal
restriction sites on the foreign
 
DNA.
 
Alternatively 
a chemically 
synthesized adaptor
molecule which have 
a 
performed 
cohesive
end 
can 
be
 
used.
A
n
 
a
d
a
p
t
o
r
i
s
 
a
 
s
h
o
r
t
,
 
c
h
e
m
i
c
a
l
l
y
s
y
n
t
h
e
s
i
z
e
d
,
 
d
o
u
b
l
e
 
s
t
r
a
n
d
e
d
 
D
N
A
m
o
l
e
c
u
l
e
w
h
i
c
h
 
i
s
 
u
s
e
d
 
t
o
 
l
i
n
k
 
t
h
e
 
e
n
d
s
 
o
f
 
t
w
o
 
o
t
h
e
r
D
N
A
 
m
o
l
e
c
u
l
e
s
.
The 
adaptor molecule have 
one 
blunt end
bearing 5’ phosphate group 
& a cohesive 
end
which 
is not
 
phosphorylated.
 
The 
use of adaptors: (a) the actual structure of an adaptor,
showing the modified 
5′-OH terminus; 
(b) conversion of 
blunt
ends 
to 
sticky 
ends 
through the attachment of
 
adaptors.
 
The 
foreign 
DNA 
plus added adaptors is then
phosphorylated at the 5’ 
termini 
and ligated into
the
 
vector.
Problems: 
sticky 
end of adaptors 
will 
binds with
each 
other 
so 
treatment with Alkaline
Phosphates.
After attachment with target……
 
treatment
Polynucleotide Kinase 
to 
add 
P–OH 
at 
5
 
prime.
The 
basic difference between an adaptor 
&
linker 
is 
that the 
former 
has 
cohesive 
ends 
&
the latter has blunt
 
ends.
 
C
L
O
N
I
N
G
 
F
O
R
E
I
G
N
 
D
N
A
 
B
Y
 
A
D
D
I
N
G
A
D
A
P
T
O
R
S
 
H
O
M
O
P
O
L
Y
M
E
R
 
T
A
I
L
I
N
G
 
Method for joining 
DNA 
molecules make 
use of
the annealing of 
complementary 
homopolymer
sequence.
Addition of oligo(dA) sequences to the 3’ ends
of one 
DNA 
molecule 
& 
oligo(dT) blocks 
to 
the
3’ end of another
 
population.
T
h
e
 
e
n
z
y
m
e
 
t
e
r
m
i
n
a
l
 
d
e
o
x
y
n
u
c
l
e
o
t
i
d
y
l
t
r
a
n
s
f
e
r
a
s
e
 
w
i
l
l
 
c
a
t
a
l
y
z
e
 
t
h
e
 
a
d
d
i
t
i
o
n
 
o
f
 
a
 
s
t
r
i
n
g
o
f
 
n
u
c
l
e
o
t
i
d
e
s
 
t
o
 
t
h
e
 
3
'
 
e
n
d
 
o
f
 
a
 
D
N
A
 
f
r
a
g
m
e
n
t
.
One 
can 
add 
a string 
of 
G's 
to 
the 3' ends of
one 
fragment and 
a string 
of 
C's 
to 
the 3' ends
of the other
 
fragment.
 
Homopolymer
tailing: 
(a )
synthesisof 
a
homopolymer
 
tail;
(b) 
construction
 
of
a 
recombinant
DNA 
molecule
from 
a 
tailed
vector 
plus 
tailed
insert 
DNA; 
(c)
repair of the
recombinant DNA
molecule.
 
A
 
DNA
 
fragment
 
Tarminal transferase
add 
poly-dT
 
nucleotide
 
Insertion
 
Restriction
 
enzyme
 
Terminal transferase
and 
poly-d 
A
 
nucleoticle
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DNA ligation involves joining DNA fragments to vectors to create new DNA or plasmids. Methods include DNA ligase, T4 ligase, and terminal deoxynucleotidyl transferase. Linkers and adaptors play a key role in DNA cloning experiments by generating sticky ends for DNA cloning. The use of adaptors allows for combining DNA molecules with different ends, enhancing molecular biology research.


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  1. LIGATION OF DNA FRAGMENTS WITH VECTORS

  2. LIGATION OF DNA FRAGMENT WITH VECTORS DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmid. There are currently three methods for joining DNA fragments in vitro. The 1st of these is DNA ligase that covalently joins the annealed cohesive ends produced by certain restriction enzymes. The 2nd depends upon the ability of DNA ligase from phage T4-infected E. coli to catalyse the formation of phosphodiester bonds.

  3. The 3rd utilizes the enzyme terminal deoxynucleotidyl transferase to synthesize homopolymeric 3 single-stranded tails at the ends of fragments. E.coli and phage T4 encode an enzyme DNA ligase -T4 enzyme requires ATP while the E. coli enzyme requires NAD+. DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligases.

  4. LINKERS & ADAPTORS Linker is a synthetic ,short and known double stranded oligonucleotides sequence. Having blunted ends on both sides and restriction sites. E.Coli DNA ligase will not catalyse blunt end ligation except under special condition. Linker can be ligated to both ends of the foreign DNA.

  5. Treatment with R.E produces sticky ends after ligation with target DNA. Sticky ends are desirable for DNA cloning experiments. One drawback is R.E. used to generate cohesive end in the linker will also cut foreign DNA at internal sites. Solution to the problem is to choose another restriction enzyme or to methylate internal restriction sites on the foreign DNA.

  6. Alternatively a chemically synthesized adaptor molecule which have a performed cohesive end can be used. An adaptor is a short, chemically synthesized, double stranded DNA molecule which is used to link the ends of two other DNA molecules. The adaptor molecule have one blunt end bearing 5 phosphate group & a cohesive end which is not phosphorylated.

  7. The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5 -OH terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors.

  8. The foreign DNA plus added adaptors is then phosphorylated at the 5 termini and ligated into the vector. Problems: sticky end of adaptors will binds with each other so treatment with Alkaline Phosphates. After attachment with target treatment Polynucleotide Kinase to add P OH at 5 prime. The basic difference between an adaptor & linker is that the former has cohesive ends & the latter has blunt ends.

  9. CLONING FOREIGN DNA BY ADDING ADAPTORS

  10. HOMOPOLYMER TAILING Method for joining DNA molecules make use of the annealing of complementary homopolymer sequence. Addition of oligo(dA) sequences to the 3 ends of one DNA molecule & oligo(dT) blocks to the 3 end of another population. The enzyme terminal deoxynucleotidyl transferase will catalyze the addition of a string of nucleotides to the 3' end of a DNA fragment. One can add a string of G's to the 3' ends of one fragment and a string of C's to the 3' ends of the other fragment.

  11. Homopolymer tailing: (a ) synthesisof a homopolymer tail; (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA; (c) repair of the recombinant DNA molecule.

  12. A DNA fragment Tarminal transferase add poly-dT nucleotide Restriction enzyme Terminal transferase and poly-d A nucleoticle Insertion

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