Restriction enzymes can cut DNA

Restriction enzymes can cut DNA : 

 Although restriction enzymes can cut DNA at precise
sequences, they can not join two DNA fragments, which is essential
to form a hybrid DNA molecule from two different sources. Joining
of two DNA molecuLes requires 5’-+3’ phosphodiester bond
formation and is an energy dependant process. Specific enzyme
called DNA ligase performs this action by using ATE’ molecule to
join two adjacent DNA molecules. Any two dsDNA fragments
having blunt ends can be joined by DNA ligase by utilizing two
ATP molecules. As blunt ends are same for all the restriction
enzymes, two fragments digested by two different blunt end
cutters can be joined easily. This universality makes blunt end
joining a popular tool in molecular cloning. However, efficiency
of blunt end joining is rather low. This is due to the fact that DNA
ligase can not bring two DNA molecules nearby; only when two
molecules are adjacent, it can catalyze the joining. This makes
blunt end ligation a random process which depends on the vicinity
of two DNA molecules in solution. For this reason blunt end ligation is preferred in joining short oligonucleotides, where concentration of free ends as well as the enzyme is quite high. Application of chemicals that can aggregate DNA or increase the concentration of the solution improves the efficiency of blunt end ligation.
         Presence of sticky ends improves ligation efficiency because
fragments with complementary overhang sequences bind to each
other securely by hydrogen bonding. DNA ligase forms the
phosphodiester bridge between the fragments more efficiently.
Difficulty in this system is that the sticky ends are restriction
enzyme specific. Both the fragments are to be cut with either same
restriction enzyme or isoschizomers, which limit the applicability
of the ligation method. To obtain the efficiency of sticky end
ligation and universality of blunt end ligation, hybrid
methodologies are used where after digestion with blunt end
cutters the blunt ends arc ligated to synthetic oligonucleotides
like linkers or adapters that bear sites for sticky ends. The hybrid
molecules then can be digested with sticky end cutters to generate
overhang structures and can be oincd more efficiently by DNA
ligase. The Iink€rs arc short DNA molecules that have restriction
sites for sticky ends, so digestion with a sticky end cutter is
necessary for generating sticky ends. Adapters, on the other hand
are synthetic DNA molecules being blunt at one end and sticky at
the other end. Therefore further digestion with a sticky end cutter
is not required alter adapter ligation and the DNA fragments can
directly be joined by DNA ligase.
      DNA can also be joined without using DNA ligase in vitro. in
a system known as homopolyrner tailing, an enzyme terminal
translerase is used that adds dNTPs to the 3’ end of DNA. Two
different fragments can be extended by adding complementary
repeatitive polynucleotidcs (poly (A) to one strand and poly (T)
to another strand) upto a certain length. The resultant fragments
will be held together by complementary base pairing and can be
directly transferred into a target cell. The cells DNA repairing
machinery ligates the fragments by phosphodiester bridge
formation.