TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.

1. T/A CLONING

2.  TA cloning is a subcloning technique that
doesn't use restriction enzymes and is easier
and quicker than traditional subcloning.
 The technique relies on the ability of adenine
(A) and thymine (T) (complementary base
pairs) on different DNA fragments to
hybridize and, in the presence of ligase
become ligated together. PCR products are
usually amplified using Taq DNA polymerase
which preferentially adds an adenine to the 3'
end of the product

3.  Such PCR amplified inserts are cloned into
linearized vectors that have complementary
3' thymine overhangs. Commercialized kits
with pre-prepared vectors and PCR reagents
are currently sold, greatly speeding up the
process.

4. PROCEDURE
 Creating the insert
Insert is created by PCR using Taq DNA
Polymerase.
This polymerase lacks 3' to 5' proofreading
activity and, with a high probability, adds a single,
3'-adenine overhang to each end of the PCR
product. It is best if the PCR primers have
guanine at the 5' end as this maximizes
probability of Taq DNA polymerase adding the
terminal adenosine overhang.

5.  Thermostable polymerases containing
extensive 3´ to 5´ exonuclease activity
should not be used as they do not leave the
3´ adenine-overhangs

6. CREATING THE VECTOR
 The target vector is linearized and cut with a
blunt-end restriction enzyme
 This vector is then tailed with ddTTP using
terminal transferase.
 (ddTTP to ensure the addition of only one T
residue)
 This tailing leaves the vector with a single 3'-
overhanging thymine residue on each blunt
end.

7. DIAGRAM OF TA CLONING

8. ADVANTAGES OF TA CLONING
 No need of restriction enzymes other than
generating the linearized vector.
 Procedure is simple and fast as compared to
traditional way of cloning.
 TA cloning can be used where no restriction
sites can be used in traditional cloning.

9. DRAWBACK
 The major problem is that the gene has a
50% chance of getting cloned in the reverse
direction.
 The method also needs DNA ligase.