How homologous do homology arms need to be? - (Jan/22/2014 )
I am going to be using homologous recombination to insert a gene into a bacterial chromosome. I have a previously constructed plasmid with my gene of interest including the homology arms. This plasmid was used successfully in a different strain of the same species. I have looked at the homology arms (500bp) in relation to my strain and there are some gaps and some base changes as follows:
Right homology arm:
5 base changes & 2 gaps. These are located in the middle of the sequence and not within the first or last 100 bases.
Left homology arm:
15 base changes & 0 gaps. These are located throughout the whole 500bp sequence one being with in the first 5 bases.
I have a feeling that this might lead to failed cloning and I have looked for information on the web but I can not find anything useful. If anyone can shed some light on this for me or point me in the direction of a good information resource I would be very grateful.
Thanks in advance
Check this: http://www.pnas.org/content/82/14/4768.full.pdf.
Its hard to predict how good it will work, but you could indeed expect problems. It also depends on what bacterium you will work with.
500bp is pretty long anyway.
Thanks for the reply. I will take a look at that paper. Unfortunately I am working with Clostridia which, as you may know, are not very happy to be genetically manipulated. They also have a low GC content (~30%). From what I have read, for homologous recombination to work in Clostridia long homology arms are required; 300 - 500bp which a negative selection marker and up to 1.2kb without.
I was just hoping someone might have some experience with this. As you can appreciate, it is not cheap to have long sequences synthesised and it's very labour intensive to produce them yourself. I have a feeling I may have to do this though.
Thanks again :)
For producing homology arms, you can simply PCR from the genomic DNA, rather than having it synthesized.