TA cloning is a cloning technique that avoids restriction enzymes. It relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize when ligated together. The technique involves using a PCR product amplified with Taq polymerase, which leaves 3' adenine overhangs, and a linearized vector with 3' thymine overhangs. The complementary overhangs allow the PCR product to ligate directly into the vector. Examples of vectors designed for TA cloning, such as pLUG vectors, were also discussed. TOPO TA cloning uses topoisomerase enzyme to ligate PCR products to vectors. Ligation independent cloning uses annealing of single-stranded overhangs of at least 12 bases

1. Strategies for cloning PCR products
Dr. Manikandan Kathirvel M.Sc., Ph.D.,
(NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Reaccredited with "A++" Grade by NAAC
K. Narayanapura, Kothanur (PO)
Bengaluru 560077
Email: manikandan@kristujayanti.com
ORCID ID: 0000000270066334

2. TA CLONING
TA cloning (also known as rapid cloning or T cloning) is a cloning technique that avoids the use of restriction
enzymes and is easier and quicker than traditional cloning.
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.

3. TA Cloning Functioning
 TA cloning is brought about by the terminal transferase activity of certain type of DNA polymerase such as
the Taq polymerase.
 PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3' end of
the product. This enzyme adds a single, 3'-A overhang to each end of the PCR product.
 As a result, the PCR product can be directly cloned into a linearized cloning vector that have single base
3'-T overhangs on each end. Such vectors are called T- vectors.
 The PCR product with A overhang, is mixed with this vector in high proportion. The complementary
overhangs of a "T" vector and the PCR product with dA overhang hybridize. The result is a recombinant
DNA, the recombination being brought about by DNA ligase.

4. Procedure
Diagram of TA Cloning
Creating the insert
The insert is created by PCR using Taq 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 guanines at
the 5' end as this maximizes probability of Taq DNA
polymerase adding the terminal adenosine overhang.
Thermostable polymerases containing extensive 3´ to 5´
exonuclease activity should not be used as they do not leave the
3´ adenine-overhangs.
Creating the vector
The target vector is linearized and cut with a blunt-end
restriction enzyme. This vector is then tailed with
dideoxythymidine triphosphate (ddTTP) using terminal
transferase. It is important to use 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.

5. Examples
pLUG® and pLUG®-Multi TA-cloning vectors were
designed for the convenient and direct PCR cloning and
serving a credible blue/white colony selection.
Features of the pLUG TA-cloning Vectors
Multiple cloning region
The multiple cloning region is located around the TA-cloning site in the pLUG
TA-cloning vectors. The restriction-enzyme recognition sites of pLUG cloning
vector around the TA-cloning site are located in mirror-repeat pattern.
Origin of replication
The pLUG TA-cloning vector has f1and ColE1origins of replication which is
responsible for the replication of plasmid.
Ampicillin resistance gene
The pLUG TA-cloning vectors have bla gene encoding for beta-lactamase that
confer resistance to ampicillin.
LacZ alpha sequence
The fragment of lacZ alpha sequence in the pLUG TA-cloning vectors is able to
complement betagalactosidase activity. The lacZ alpha sequence reduces the time
to screen for positive clones.
M13 forward / reverse priming sites
The M13 forward/reverse priming sites in the pLUG TA-cloning vectors allow
convenient sequencing.

6. TOPO TA cloning of Taq-amplified DNA

7. TOPO –TA cloning
 The TA TOPO cloning technique relies on the ability of adenine (A) and
thymine (T) (complementary basepairs) on different DNA fragments
to hybridize and, in the presence of ligase or topoisomerase, become
ligated together.
 The insert is created by PCR using Taq DNA polymerase, a polymerase
that lacks 3' to 5' proofreading activity and with a high probability adds a
single, 3'-adenine overhang to each end of the PCR product.
 Cloning fragments amplified by either Taq or Pfu polymerase as Taq
polymerase (unlike Pfu) leaves an extra "A" nucleotide at the 3'end
during amplification.
 The key to TOPO cloning is the enzyme DNA topoisomerase I, which
functions both as a restriction enzyme and as a ligase.
 Its biological role is to cleave and rejoin DNA during replication.
 Vaccinia virus topoisomerase I specifically recognizes the pentameric
sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate
group attached to the 3´ thymidine.
 It cleaves one DNA strand, enabling the DNA to unwind. The enzyme
then religates the ends of the cleaved strand and releases itself from the
DNA.
 To harness the religating activity of topoisomerase, TOPO vectors are
provided linearized with topoisomerase I covalently bound to each 3´
phosphate.
 This enables the vectors to readily ligate DNA sequences with compatible
ends (Figures 1, 2).
 The ligation is complete in only 5 minutes at room temperature.
Figure 1. TOPO TA cloning of Taq-amplified DNA.
"Sticky end" TOPO TA cloning

8. Procedure:
 TOPO vectors are designed in such a way that they carry
this specific sequence 5´-(C/T)CCTT-3' at the two linear
ends.
 The linear vector DNA already has the topoisomerase
enzyme covalently attached to both of its strands' free 3'
ends. This is then mixed with PCR products.
 When the free 5' ends of the PCR product strands attach to
the topoisomerase/3' end of each vector strand, the strands
are covalently linked by the already bound topoisomerase.
 This reaction proceeds efficiently when this solution is
incubated at room temperature with required salt.

9. Ligation Independent Cloning (LIC)
LIC is a cloning method that makes use of annealing of single-stranded complementary overhangs on the target vector and
a PCR-generated insert of at least 12 bases overlap region.
Single-stranded overhangs can be generated by using T4 DNA polymerase and only one dNTP in the reaction mix, leading
to an equilibrium of 3'->5'-exonuclease and 5'->3'-polymerase activity at the site of the first occurrence of this nucleotide.
The incubation is done with T4 DNA pol. for 30 min. and stopped by adding dCTP to the reaction mix.
After annealing of vector and insert, the mixture is used to transform E. coli.