TOPO Cloning

TOPO Cloning is a molecular biology technique in which DNA fragments amplified by either Taq or Pfu polymerases are cloned into specific vectors without the requirement for DNA ligases.

Principle

The technique utilises the inherent biological activity of DNA topoisomerase I. The biological role of topoisomerase is to cleave and rejoin supercoiled DNA ends to facilitate replication. Vaccinia virus topoisomerase I specifically recognises DNA sequence 5´-(C/T)CCTT-3'. During replication, the enzyme digests DNA specifically at this sequence, unwinds the DNA, re-ligates it again at the 3' phosphate group of the thymidine base.

Technique

TOPO vectors are designed in such a way that they carry this specific sequence 5´-(C/T)CCTT-3' at the two linear ends. This is then mixed with PCR products. The PCR products move align themselves between the linear ends of the vector. This alignment is covalently linked by the already bound topoisomerase when this solution is incubated at room temperature with required salt. [ [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/PCR-cloning/PCRC-Misc/The-Technology-Behind-TOPO-Cloning.html Invitrogen website explaining the TOPO cloning principle] ] Different type of vectors are used for cloning fragments amplified by Taq or Pfu polymerase as Taq polymerase unlike Pfu polymerase leave an extra nucleotide "A" at the 3'end during amplification.

References