TOPO cloning: Difference between revisions
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== Technique == |
== Technique == |
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TOPO vectors are designed in such a way that a vector carrying this specific sequence 5´-(C/T)CCTT-3 sequence at the two linear ends are mixed with PCR products. The PCR products move themselves to align between the linear ends of the vector. This alignment is covalently linked by the already bounf topoisomerase when this solution is incubated at room temperature with salt sollution. |
TOPO vectors are designed in such a way that a vector carrying this specific sequence 5´-(C/T)CCTT-3 sequence at the two linear ends are mixed with PCR products. The PCR products move themselves to align between the linear ends of the vector. This alignment is covalently linked by the already bounf topoisomerase when this solution is incubated at room temperature with salt sollution.<ref>[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]</ref> |
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== References == |
== References == |
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Revision as of 04:48, 20 June 2008
TOPO Cloning is a procedure by 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 amazing potential of DNA topoisomerase I. The biological role of topoisomerase is to cleave and rejoin supercoiled DNA ends to facilitate replication. Vaccini 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, religates the DNA again at the 3' phosphate group of the thymidine base.
Technique
TOPO vectors are designed in such a way that a vector carrying this specific sequence 5´-(C/T)CCTT-3 sequence at the two linear ends are mixed with PCR products. The PCR products move themselves to align between the linear ends of the vector. This alignment is covalently linked by the already bounf topoisomerase when this solution is incubated at room temperature with salt sollution.[1]