4 fragment ligations.....?

[Ned Mantei]

In article <39A14326.D7814A9F at biocomp.unl.edu>, Chris LaRosa 
<clarosa at biocomp.unl.edu> wrote:

>Recently there was a discussion on the feasiblility of complex
>ligations.
>I am contemplating attempting a 4 dna fragment ligation.  The constuct
>will be directional with all sticky ends.   Any comments or references
>would be appreciated. 

I have done ligations with as many as 5 fragments (vector, 3 fragments, 
double-stranded olio). The conditions you mention (directional, all 
sticky ends) very much favor success.
Things I considered in my own constructs:
1) All pairs of ends were different, so that the fragments could only 
fit together in one way. 
2) There was only one pair of blunt ends. I would not attempt a many-way 
ligation with more than that, as the efficiency is too low.
3) For a 5-way ligation, the frequency of correct clones is rather low. 
In some cases I did a colony lift and hybridization to find the few % of 
colonies with the main insert fragment. This shouldn't be necessary for 
a 4-way ligation.
4) There can be problems with contamination from vectors from which the 
various insert fragments were isolated. When isolating insert fragments, 
I often cut with additional enzymes so that the (contaminating) vector 
couldn't recircularize. 
5) Even if the final vector had two different ends, I treated it with 
phosphatase.

Finally, if I had such projects to do again, I would consider the method 
described by Pachuk, et al., Gene 243:19-25, 2000, "Chain reaction 
cloning: a one-step method for directional ligation of multiple DNA 
fragments". The method seems very clever, but I haven't tried it myself.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland