TOPO cloning

Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products.

[1] The vectors in commercially available TOPO kits have been added to the topoisomerase site embedded in a beta-galactosidase cassette allowing blue-white scanning.

The vector ends thus self-assemble, resulting in the production of blue colonies that do not need to be selected and sequenced for potential positive clones.

[1] The TA TOPO cloning 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 or topoisomerase, become ligated together.

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.