TA Cloning

TA cloning involves base pairing of adenine (A) and thymine (T)  (complementary base pairs) that result in the joining of DNA fragments to the linearized cloning vector. This strategy is very useful as it allows direct cloning of PCR-amplified DNA fragments to T-vectors without any use of restriction enzymes (Green & Sambrook, 2021; Zhou & Gomez-Sanchez, 2000).

The cloning vector contains an extra T as a 3’-overhang and is prepared by enzymatic reactions that can involve either Terminal transferase enzyme or Taq DNA Polymerase (non-proofreading). Some restriction enzymes such as XcmI (Kovalic et al., 1991), HphI, and MboII can also generate a 3’- T overhang and can be used for preparing T-vector provided their recognition site is present in multiple cloning site of the cloning vector and the digestion results in just linearization of the vector. 

DNA fragments that are to be cloned usually are the products of polymerase chain reaction that are performed in presence of non-proofreading thermostable DNA polymerase (e.g., Taq). The property of taq DNA polymerase to incorporate an extra nucleotide, preferentially an adenosyl (A) residue, at the 3’-end results in DNA fragments that contain “A” overhang at 3’-termini. 

When these two DNA molecules are incubated in presence of T4 DNA ligase enzyme in presence of suitable buffer and incubation conditions, “A” overhang of DNA fragments and “T” overhang (sticky ends) of cloning vectors base pair and subsequently their ends are ligated by T4 DNA Ligase, resulting in a single DNA molecule. Due to T-overhand at 3’-termini, self ligation of vector remains low, resulting in high efficiency of cloning.

Advantages:

• Relatively easy and fast (as compared to blunt end cloning)
• Direct cloning of PCR amplified fragments without a need of digesting it with restriction enzymes

Disadvantages:

• Non-directional cloning (orientation of DNA fragment can not be controlled)

REFERENCES

• Kovalic et al., 1991. General method for direct cloning of DNA fragments generated by the polymerase chain reaction. Nucleic Acids Res. 19 (16), 4560. PMID-1886782; Full-Text Link: academic.oup, PMC328657
• Green & Sambrook, 2021. Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning. Cold Spring Harb Protoc. 2021(6). PMID-34074739; Full Text Link: cshprotocols (download PDF)
• Zhou & Gomez-Sanchez, 2000. Universal TA cloning. Curr Issues Mol Biol. 2(1), 1-7. PMID-11464915; Full-Text Links: caister (download PDF)