Topo cloning

Learn about topo cloning with this video.
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TOPO cloning is a molecular biology technique in which DNA fragments amplified by Taq polymerase are cloned into specific vectors without the requirement for DNA ligases. The Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosin (A) to the 3'-end of the PCR products. This characteristic is exploited in TOPO-cloning.
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 and re-ligates it again at the 3' phosphate group of the thymidine base.
TOPO vectors are designed in such a way that they carry this specific sequence 5´-(C/T)CCTT-3' at the two linear ends. The linear vector DNA already has the topoisomerase enzyme covalently attached to both of its strands' free 3' ends. This is then mixed with PCR products. When the free 5' ends of the PCR product strands attack the topoisomerase/3' end of each vector strand, the strands are covalently linked by the already bound topoisomerase. This reaction proceeds efficiently when this solution is incubated at room temperature with required salt.[1] Different types of vectors are used for cloning fragments amplified by either Taq or Pfu polymerase as Taq polymerase (unlike Pfu) leaves an extra "A" nucleotide at the 3'end during amplification.
The TA TOPO cloning technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase or topoisomerase, become ligated together. The insert is created by PCR using Taq DNA polymerase, a polymerase that lacks 3' to 5' proofreading activity and with a high probability adds a single, 3'-adenine overhang to each end of the PCR product. 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. Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity should not be used as they do not leave the 3´ adenine-overhangs. The target vector is linearized and cut with a blunt-end restriction enzyme. This vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. It is important to use ddTTP to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end.

[@geronzyx910]

The most comprehensive video on the subject I have seen ! It really helped me understand thank you !

[@shomusbiologyofficial]

Glad to hear that you're getting benefit from my lectures

[@neurobiogirl]

THANK YOU!! I have been watching your lectures today. You are a awesome teacher!!

[@dabram1707]

you want to listen only about TOPO just jump 6:30 :)

[@GirishKumar-lb9oo]

Thank you

[@alanoudtalalalgasham7964]

you don't have idea how you helped me. I really appreciated. good job and thank you so much

[@jared394]

amazing job, not an easy technique to explain you and you nailed it!

[@128scarface]

Simple yet very informative, thank you very much !!

[@lauragoo9792]

This is explained so well! Thank you so much you just saved my life.

[@shomusbiologyofficial]

+Laura Goo glad you liked my lectures

[@Federchen]

Thank you so much for this great and detailed explanation!

[@shomusbiologyofficial]

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[@118lock]

thank you so much! you explained it very well!

[@mayukhroy5393]

Sir, you made all my doubts clear.

[@shomusbiologyofficial]

You're welcome. Glad to hear that you're getting benefit from my lectures

[@vanessaong4421]

Omg you helped me so much!! Amazing video!

[@user-rd9js8jo3o]

But can it be ligated by TOPO without insert?

[@saro4761]

Fantastic video!! Thanks so much...

[@kankanabanerjee7541]

Thanks a lot! Great explanation !!

[@tariqmahmood3391]

VERY GOOD EXPLAINED

[@AnkushSharma-zv5hv]

very well explained

[@wtharani96]

Thank you somuch for your explanation😍

[@shomusbiologyofficial]

You're welcome. Glad to hear that you're getting benefit from my lectures

[@pramitbanerjee]

i understood it! thanks!

[@miyssaimam3228]

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[@shomusbiologyofficial]

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[@racooninabag]

well played fam

[@fathimasalim580]

Ohh god! Thank you

[@shomusbiologyofficial]

You're welcome