TA cloning (also known as rapid cloning or T cloning) is a

subcloning In molecular biology, subcloning is a technique used to move a particular DNA sequence from a ''parent vector'' to a ''destination vector''. Subcloning is not to be confused with molecular cloning, a related technique. Procedure Restriction ...

technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. The technique relies on the ability of

adenine Adenine (, ) (nucleoside#List of nucleosides and corresponding nucleobases, symbol A or Ade) is a purine nucleotide base that is found in DNA, RNA, and Adenosine triphosphate, ATP. Usually a white crystalline subtance. The shape of adenine is ...

(A) and

thymine Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...

(T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of

ligase In biochemistry, a ligase is an enzyme that can catalyze the joining ( ligation) of two molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the molecules, typically resulting i ...

, become ligated together. PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3' end of the product. Such PCR amplified inserts are cloned into linearized vectors that have complementary 3'

overhangs.

Procedure

Creating the insert

The insert is created by PCR using

Taq polymerase ''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by master's student Alice Chien et al. in 1976. Its name is often abbr ...

. This polymerase lacks 3' to 5' proofreading activity and, with a high probability, adds a single, 3'-

overhang to each end of the PCR product. It is best if the PCR primers have

guanine Guanine () (symbol G or Gua) is one of the four main nucleotide bases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine ( uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside ...

s 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. Manufacturers commonly sell TA Cloning "kits" with a wide range of prepared vectors that have already been linearized and tagged with an overhanging thymine.

Benefits and drawbacks

Given that there is no need for restriction enzymes other than for generating the linearized vector, the procedure is much simpler and faster than traditional

. There is also no need to add restriction sites when designing primers and thus shorter primers can be used saving time and money. In addition, in instances where there are no viable restriction sites that can be used for traditional cloning, TA cloning is often used as an alternative. The major downside of TA cloning is that directional cloning is not possible, so the gene has a 50% chance of getting cloned in the reverse direction.

Procedure

Creating the insert

Creating the vector

Benefits and drawbacks

References

See also