Homologous Recombination - (Oct/06/2005 )

Hi
I am trying to knockout the gene of interest using the PCR fusion strategy but am having problems with getting the plasmid inserted into the genome for the first cross over event. My fusion fragment has more than 300 bp on one side and i was wondering if these many bps are enough for recombination to take place.

Thanks

It really depends on the organism you're trying to cross it into. We use this technique routinely to create mutations in B. fragilis, and have found that (in our hands, at least), we need > 500 bp for each flank for the suicide vector to cross in efficiently. After making hundreds of such mutations over the years, we don't even think about designing such an experiment without allowing > 1 kb for each flank.

I should explain a bit -- what we do is PCR up DNA flanking each side of the desired mutation, put a restriction site for cloning on each of the two outer primers, and a different restriction site on the two inner primers. After PCR, digestion, and gel cleanup of the two flanks, we ligate them together with digested and CIP treated vector in a three-way ligation, and move this suicide construct into the target strain. On cross out, the deletion engineered between the two flanks remains (hopefully).

We've also found it very important to balance the GC content of the flanking DNA. If the flanks differ in GC content, say >5% difference, increase the size of the AT-rich flank by 300 - 500 bases.

For A. calcoaceticus, the magic number is >500. For some other organisms, it's >250.

It really depends on your organism.

-Matt