Topo Cloning - Understanding the principle (Jan/29/2011 )

Hi!

I'm not sure if I understand the principle of the "Topo Cloning" correctly, so I hope that
some can tell me if I'm right or wrong:

- the vector is linear and is looking somewhat like this
3' Topo-P-T-------------------5'
5' ------------------T-P-Topo 3'

Here there is the topoisomerase enzyme covalently bound to a phosphate of a thymidin resudue at the
3' end

Question: Are there also a free phosphate residues on the 5' ends?

- because a free 3'-OH end is missing the vector can't ligate itself to become circular

- if I add my PCR-insert with the free 3'OH ends the topoisomerase will use these OH groups
to connect vector and insert

Question: If the 3'-P-Topo end of my vector connects to the 5'-P end of the insert what will
happen to the second phosphate?
I could imagine that it could be used to connect the 5' phosphate free end of the
vector to the 3' of the insert!?

I hope someone can give me some good explanations on how that method exactly works

br,
Ikar

See here for a good summary.

Thanks. But unfortunately this summary does not give answers to my question about the 5'-phosphate.

I'm pretty sure the the phosphates are used to supply the energy for making the ligation between the insert and vector.

bob1 on Mon Jan 31 22:24:25 2011 said:

Ok, you think that there are phosphates on both, phsophates on the 3' ends of the vector and also at the 5' ends of the vector DNA?

I don't think I understand what you are trying to say... you realise that these clonings are double stranded right, and that the phosphate ends can't be ligated together?

Sorry for my confused questions...

I've found a nice webpage which explains the principle
quite good.

http://bioinforx.com/lims/bxseqtools/ultimate-molecular-cloning-guides/Directional-TOPO-Cloning

I think that I've understood the principle now.