Question

What makes TOPO cloning directional?

1) The presence of a 5' overhang on the vector and a 3' overhang on the insert
2) The presence of a 3' overhang on the vector and a 5' overhang on the insert
3) The presence of a 5' overhang on both the vector and the insert
4) The presence of a 3' overhang on both the vector and the insert

Answer 1

TOPO cloning is directional because it uses a vector with 5' overhangs and an insert with 3' overhangs, ensuring that the insert can only ligate into the vector in one direction.

Step-by-step explanation:

TOPO cloning is considered directional cloning due to the presence of a 5' overhang on the vector and a 3' overhang on the insert. In TOPO cloning, the enzyme topoisomerase recognizes and binds to the overhanging ends of the DNA. The vector is engineered to have a single, overhanging (sticky) end at each 5' end, while the PCR-generated insert has a complimentary overhang at each 3' end. This setup ensures that the insert can only ligate into the vector in one direction because the overhangs are not compatible with the opposite ends, and thus, ligation of the insert in the reverse direction is not possible. By employing the topoisomerase I enzyme which cleaves and rejoins DNA ends, and stratagem of complementary overhanging ends that are unidirectional, TOPO cloning facilitates the rapid and efficient insertion in the desired orientation.