RFD: Plasmid re-ligation without ligase?

[Jennifer Lyon]

In <5jipvu$jhd$2 at sparcserver.lrz-muenchen.de>
salger at wap1.zi.biologie.uni-muenchen.de (Klaus Salger) writes: 

>as far as I remember I read about a system called "Ligation
>Independent Cloning System". I don't remember the details but I think
>it relies on in vivo ligation (and recombination?).

In Ligation Independent Cloning, you use a much longer overhang than
traditional restriction site cloning. Novagen's LIC vectors have 12-14
bp overhangs. Special primers (which include the 'LIC site') are used
to PCR-amplify inserts, which are then treated with T4 DNA polymerase
in the presence of only one dNTP so that the polymerase chews back
until it hits a specific site and then pops in the one available
nucleotide and stops digesting. This leaves you with a 12-14bp overhang
complimentary to the one in the prepared vector. Insert and vector are
then annealed to each other, resulting in a complex that is stable
enough to be transformed effectively into e. coli. The breaks are far
enough apart that they are treated as single-strand nicks and are
'healed' (re-ligated) within the e. coli cells. So in effect, this does
involve in vivo ligation, and it works quite well.

-Jenny
Novagen Technical Services
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