Question

Is TOPO cloning technique directional?  Describe what is meant by “directional”, and how is this achieved.

Answer 1

Directional cloning is an efficient method to direct the insert into the vector in the right orientation. This cloning is used to maintain correct open reading frame for protein expression. It can also be used to maintain proper position for cis acting regulatory elements. It is performed by first digesting the insert and vector with the specific two restriction enzymes. This creates blunt or sticky ends which can then be used to ligate the vector and insert in the right orientation. As the 5’ and 3’ sticky ends are cleave with specific restriction sites both on vector and insert, the orientation of the insert will be only in one direction. The 5’ and 3’ sticky ends are non-complementary to each other as they are cut by different restriction enzymes.

Normal TOPO cloning is not directional cloning. It uses Topoisomerase 1 as the restriction enzyme. This enzyme recognized pentameric sequence 5´-(C/T)CCTT-3´. It then forms a covalent bond with the phosphate group of 3’ thymine. Cleavage of one strand of DNA causes it to unwind. The isomerase will then relegates to end of strand and are released from the DNA.

Directional TOPO cloning can be however be performed. It uses a 5’ GTGG overhang on the 5´ end. There is also a blunt end on the 3´ end in the vector. This vector anneals with the CACC sequence included in primer. The DNA ligase can then seal the vector and insert in right orientation.